JP7549306B2 - Development of novel phospholipids and their applications, as well as methods for separating and measuring phospholipids - Google Patents

Description

The present invention relates to a novel phospholipid and its use. Furthermore, the present invention relates to a method for separating and measuring phospholipids. More specifically, the present invention relates to a technique for identifying the position of the phosphate group of phosphatidylinositol phosphate and/or lysophosphatidylinositol phosphate while keeping the acyl group intact. In particular, the present invention provides a method for separating and measuring phospholipids using mass spectrometry (MS) optionally in combination with chromatography or ion mobility separation (IMS).

Lipids are essential components of life as membrane constituents, energy sources, and signaling molecules. Among lipids, phospholipids in particular are an important group of molecules that regulate a variety of cellular functions by targeting intracellular proteins to cell membranes and intracellular organelles and acting as spatiotemporal regulators to control their activity at that location. However, because they are poorly soluble in water and are not directly controlled by genes, there are limitations to their analysis, making them a relatively underexplored area of biochemistry.

The present inventors have taken on the challenge of this unexplored field by using new techniques to generate genetically modified mice with phospholipid metabolic enzymes and phospholipid-binding domains. The present inventors' research has become a global pioneer, and the elucidation of the biological function regulation mechanism by intracellular phospholipids has progressed to the extent that it can be reconsidered as a new research field. In other words, the association between various pathologies such as various cancers, allergies, hepatic steatosis/fibrosis, cardiac dysfunction, neurodegeneration, and enteritis and abnormalities in individual phospholipid metabolic enzymes has been discovered one after another, and there are high expectations for future developments as basic research results that can lead to the development of new medicines (Non-Patent Document 1).

On the other hand, research to date has focused on metabolic enzymes to which genetic techniques can be applied, and little progress has been made in research into the changes in phospholipids themselves (Non-Patent Document 1). This is because methods for detecting, identifying, and quantifying lipids have yet to be developed. Unlike genes, the types and numbers of lipids that make up living organisms are still unclear, and it is believed that many unknown lipids lie dormant and undiscovered.

In addition, it is known that abnormal expression of phospholipid metabolic enzymes is related to pathologies such as cancer and metabolic syndrome, but the details of the molecular mechanism are unknown. There are eight classes of phosphoinositides (also called phosphatidylinositol phosphate, phosphatidylinositol phosphoric acid (salt), etc.) that are involved in many diseases (phosphatidylinositol, phosphatidylinositol-3-monophosphate, phosphatidylinositol-4-monophosphate, phosphatidylinositol-5-monophosphate, phosphatidylinositol-3,4-diphosphate, phosphatidylinositol-3,5-diphosphate, phosphatidylinositol-4,5-diphosphate, and phosphatidylinositol-3,4,5-triphosphate). Each class of these lipids or metabolic products may have different roles and functions, and their metabolic enzymes and receptors may be significant as targets for therapeutic drugs for various diseases and as disease markers.

Sasaki T. et al. , Mammalian phosphoinositide kinases and phosphotases. Prog Lipid Res. 2009 Nov;48(6):307-43, doi:10.1016/j. pli pres. 2009.06.001 Kielkowska, A. , et al. A new approach to measuring phosphoinositides in cells by mass spectrometry. Adv Biol Reql 54, 131-141 (2014).

In view of the above, the present inventors have embarked on the development of a new lipid analysis method based on mass spectrometry. The advantages of using mass spectrometry are that it is possible to analyze biological samples (human clinical specimens, etc.) that could not be analyzed without using radioisotope labels, and that it is possible to distinguish the composition of acyl groups (hence, it is possible to discover the lysophospholipids of the present invention). A new method for measuring phosphatidylinositol phosphates (also referred to as "PIPs" in this specification), which are particularly difficult to measure because they are present in small amounts in the body and are subject to multivalent phosphorylation, has been established. Using this technology, it has been possible to discover unknown lysophosphatidylinositol phosphates (also referred to as "LPIPs" in this specification) that had been impossible to identify until now. Since there were no methods for identifying, detecting, or synthesizing LPIPs until the disclosure of the present invention, the disclosure of the present invention is the first report of LPIPs as a substance.

The measurement method of the present invention was completed by the unexpected discovery that phosphoinositides and lysophosphoinositides (also referred to as lysophosphatidylinositol phosphate, lysophosphatidylinositol phosphoric acid (salt), etc.) can be measured, detected, and identified by specifying the position of the phosphate group in the state having an acyl group (also referred to as "intact state" in this specification) using chromatography-mass spectrometry. In particular, the present invention provides the first method for detecting and identifying all eight classes of phosphoinositides and all eight classes of lysophosphoinositides having information on the acyl group (fatty acid side chain) using liquid column chromatography/mass spectrometry (LC-MS) or ion mobility separation-mass spectrometry (IMS-MS). The columns used here can include, but are not limited to, chiral columns, hydrophobic columns, etc., and any chromatography that separates hydrophilic and hydrophobic groups can be used.

Thus, the present invention provides the following:
(Item 1A)
A compound which is lysophosphatidylinositol monophosphate, lysophosphatidylinositol diphosphate or lysophosphatidylinositol triphosphate.
(Item 2A)
The compound according to item 1A, which is 1-acyl-lysophosphatidylinositol monophosphate, 2-acyl-lysophosphatidylinositol monophosphate, 1-acyl-lysophosphatidylinositol diphosphate, 2-acyl-lysophosphatidylinositol diphosphate, 1-acyl-lysophosphatidylinositol triphosphate, or 2-acyl-lysophosphatidylinositol triphosphate.
(Item 3A)
1-acyl-lysophosphatidylinositol-3-monophosphate, 1-acyl-lysophosphatidylinositol-4-monophosphate, 1-acyl-lysophosphatidylinositol-5-monophosphate, 2-acyl-lysophosphatidylinositol-3-monophosphate, 2-acyl-lysophosphatidylinositol-4-monophosphate, 2-acyl-lysophosphatidylinositol-5-monophosphate, 1-acyl-lysophosphatidylinositol-3.4-diphosphate, 1-acyl-lysophosphatidylinositol The compound according to item 1A or 2A, which is 1-acyl-lysophosphatidylinositol-3.5-diphosphate, 1-acyl-lysophosphatidylinositol-4.5-diphosphate, 2-acyl-lysophosphatidylinositol-3,4-diphosphate, 2-acyl-lysophosphatidylinositol-3,5-diphosphate, 2-acyl-lysophosphatidylinositol-4,5-diphosphate, 1-acyl-lysophosphatidylinositol-3,4,5-triphosphate, or 2-acyl-lysophosphatidylinositol-3,4,5-triphosphate.
(Item 4A)
The compound according to any one of items 1A to 3A, which is 2-acyl-lysophosphatidylinositol monophosphate, 2-acyl-lysophosphatidylinositol diphosphate, 1-acyl-lysophosphatidylinositol triphosphate or 2-acyl-lysophosphatidylinositol triphosphate.
(Item 5A)
1-butanyl-lysophosphatidylinositol 4-monophosphate;
1-Hexanyl-lysophosphatidylinositol 4-monophosphate;
1-octanyl-lysophosphatidylinositol 4-monophosphate;
1-Hexadecanyl-lysophosphatidylinositol 4-monophosphate;
1-9Z-hexadecenyl-lysophosphatidylinositol 4-monophosphate;
1-heptadecanyl-lysophosphatidylinositol 4-monophosphate;
1-Octadecanyl-lysophosphatidylinositol 4-monophosphate;
1-9Z-octadecenyl-lysophosphatidylinositol 4-monophosphate;
1-9Z,12Z-octadecanedienyl-lysophosphatidylinositol 4-monophosphate;
1-8Z,11Z,14Z-icosatrienyl-lysophosphatidylinositol 4-monophosphate;
1-5Z,8Z,11Z,14Z-icosatetraenyl-lysophosphatidylinositol 4-monophosphate;
1-7Z,10Z,13Z,16Z-docosatetraenyl-lysophosphatidylinositol 4-monophosphate;
1-7Z,10Z,13Z,16Z,19Z-docosapentaenyl-lysophosphatidylinositol 4-monophosphate;
1-4Z,7Z,10Z,13Z,16Z,19Z-docosahexaenyl-lysophosphatidylinositol 4-monophosphate;
2-butanyl-lysophosphatidylinositol 4-monophosphate;
2-Hexanyl-lysophosphatidylinositol 4-monophosphate;
2-octanyl-lysophosphatidylinositol 4-monophosphate;
2-Hexadecanyl-lysophosphatidylinositol 4-monophosphate;
2-9Z-hexadecenyl-lysophosphatidylinositol 4-monophosphate;
2-heptadecanyl-lysophosphatidylinositol 4-monophosphate;
2-octadecanyl-lysophosphatidylinositol 4-monophosphate;
2-9Z-octadecenyl-lysophosphatidylinositol 4-monophosphate;
2-9Z,12Z-octadecanedienyl-lysophosphatidylinositol 4-monophosphate;
2-8Z,11Z,14Z-icosatrienyl-lysophosphatidylinositol 4-monophosphate;
2-5Z,8Z,11Z,14Z-icosatetraenyl-lysophosphatidylinositol 4-monophosphate;
2-7Z,10Z,13Z,16Z-docosatetraenyl-lysophosphatidylinositol 4-monophosphate;
2-7Z,10Z,13Z,16Z,19Z-docosapentaenyl-lysophosphatidylinositol 4-monophosphate;
2-4Z,7Z,10Z,13Z,16Z,19Z-docosahexaenyl-lysophosphatidylinositol 4-monophosphate;
1-butanyl-lysophosphatidylinositol 4,5-bisphosphate;
1-Hexanyl-lysophosphatidylinositol 4,5-bisphosphate;
1-octanyl-lysophosphatidylinositol 4,5-bisphosphate;
1-Hexadecanyl-lysophosphatidylinositol 4,5-bisphosphate;
1-9Z-hexadecenyl-lysophosphatidylinositol 4,5-bisphosphate;
1-heptadecanyl-lysophosphatidylinositol 4,5-bisphosphate;
1-Octadecanyl-lysophosphatidylinositol 4,5-bisphosphate;
1-9Z-octadecenyl-lysophosphatidylinositol 4,5-bisphosphate;
1-9Z,12Z-octadecanedienyl-lysophosphatidylinositol 4,5-bisphosphate;
1-6Z,9Z,12Z-octadecanedienyl-lysophosphatidylinositol 4,5-bisphosphate;
1-8Z,11Z,14Z-icosatrienyl-lysophosphatidylinositol 4,5-bisphosphate;
1-5Z,8Z,11Z,14Z-icosatetraenyl-lysophosphatidylinositol 4,5-bisphosphate;
2-butanyl-lysophosphatidylinositol 4,5-bisphosphate;
2-Hexanyl-lysophosphatidylinositol 4,5-bisphosphate;
2-octanyl-lysophosphatidylinositol 4,5-bisphosphate;
2-Hexadecanyl-lysophosphatidylinositol 4,5-bisphosphate;
2-9Z-hexadecenyl-lysophosphatidylinositol 4,5-bisphosphate;
2-heptadecanyl-lysophosphatidylinositol 4,5-bisphosphate;
2-octadecanyl-lysophosphatidylinositol 4,5-bisphosphate;
2-9Z-octadecenyl-lysophosphatidylinositol 4,5-bisphosphate;
2-9Z,12Z-octadecanedienyl-lysophosphatidylinositol 4,5-bisphosphate;
2-6Z,9Z,12Z-octadecanedienyl-lysophosphatidylinositol 4,5-bisphosphate;
2-8Z,11Z,14Z-icosatrienyl-lysophosphatidylinositol 4,5-bisphosphate;
2-5Z,8Z,11Z,14Z-icosatetraenyl-lysophosphatidylinositol 4,5-bisphosphate;
1-butanyl-lysophosphatidylinositol 3,4,5-triphosphate;
1-octanyl-lysophosphatidylinositol 3,4,5-triphosphate;
1-Hexadecanyl-lysophosphatidylinositol 3,4,5-triphosphate;
1-9Z-hexadecenyl-lysophosphatidylinositol 3,4,5-triphosphate;
1-heptadecanyl-lysophosphatidylinositol 3,4,5-triphosphate;
1-Octadecanyl-lysophosphatidylinositol 3,4,5-triphosphate;
1-9Z-octadecenyl-lysophosphatidylinositol 3,4,5-triphosphate;
1-5Z,8Z,11Z,14Z-icosatetraenyl-lysophosphatidylinositol 3,4,5-triphosphate;
1-4Z,7Z,10Z,13Z,16Z,19Z-docosahexaenyl-lysophosphatidylinositol 3,4,5-triphosphate;
2-butanyl-lysophosphatidylinositol 3,4,5-triphosphate;
2-octanyl-lysophosphatidylinositol 3,4,5-triphosphate;
2-Hexadecanyl-lysophosphatidylinositol 3,4,5-triphosphate;
2-9Z-hexadecenyl-lysophosphatidylinositol 3,4,5-triphosphate;
2-heptadecanyl-lysophosphatidylinositol 3,4,5-triphosphate;
2-octadecanyl-lysophosphatidylinositol 3,4,5-triphosphate;
2-9Z-octadecenyl-lysophosphatidylinositol 3,4,5-triphosphate;
2-5Z,8Z,11Z,14Z-icosatetraenyl-lysophosphatidylinositol 3,4,5-triphosphate; and 2-4Z,7Z,10Z,13Z,16Z,19Z-docosahexaenyl-lysophosphatidylinositol 3,4,5-triphosphate,
A compound according to any one of items 1A to 4A.
(Item 6A)
A composition comprising a compound according to any one of claims 1A to 5A for use as a disease marker.
(Item 7A)
The composition of claim 6A, wherein the disease marker is an inflammation marker or a cancer marker.
(Item 8A)
The composition of claim 7A, wherein the cancer comprises prostate cancer.
(Item 9A)
A method for producing the compound according to any one of items 1A to 5A, comprising the step of deacylating phosphatidylinositol monophosphate, phosphatidylinositol diphosphate or phosphatidylinositol triphosphate by contacting it with an alkylamine.
(Item 10A)
The method according to claim 9A, wherein the alkylamine is methylamine.
(Item 11A)
The method according to Item 9A or 10A, wherein the deacylation is carried out under milder conditions than the dediacylation conditions.
(Item 12A)
The method according to Item 11A, wherein the mild conditions are achieved by shortening the reaction time, reducing the concentration of the alkylamine, reducing the reaction temperature, or a combination thereof among the dediacylation conditions.
(Item 13A)
The method according to Item 11A or 12A, wherein the mild conditions are: use of methylamine as the alkylamine; a reaction time of 10 minutes or less; or a concentration of the methylamine of about 11% or less; and a reaction temperature of 53° C. or less; or a combination of the reaction time, the concentration, and the reaction temperature.
(Item 14A)
(A) providing a phosphatidylinositol monophosphate, phosphatidylinositol diphosphate, or phosphatidylinositol triphosphate having acyl groups of different masses at positions 1 and 2, wherein the acyl group having the desired mass is present at a desired position selected from positions 1 or 2;
The method for producing lysophosphatidylinositol monophosphate, lysophosphatidylinositol diphosphate, or lysophosphatidylinositol triphosphate having a desired acyl group at a desired position includes the steps of: (B) deacylating the phosphatidylinositol monophosphate, phosphatidylinositol diphosphate, or phosphatidylinositol triphosphate by contacting it with an alkylamine; and (C) as necessary, isolating lysophosphatidylinositol monophosphate, lysophosphatidylinositol diphosphate, or lysophosphatidylinositol triphosphate having the desired mass of acyl groups.
(Item 15A)
concentrating the phospholipids by contacting the sample with an anion exchange resin;
protecting the phosphate groups in the acidic phospholipids with protecting groups; and detecting, identifying or quantifying lysophosphatidylinositol phosphates in the phospholipids by mass spectrometry.
The present invention relates to a method for detecting, identifying or quantifying lysophosphatidylinositol phosphate, comprising the steps of:
(Item 16A)
The method according to claim 15A, further comprising detecting, identifying or quantifying phosphatidylinositol phosphates in the phospholipids.
(Item 17A)
The method of any one of claims 15A to 16A, wherein the protecting group comprises an alkyl group.
(Item 18A)
The method of any one of claims 15A to 17A, wherein the protecting group comprises a methyl group.
(Item 19A)
The method according to any one of claims 15A to 18A, wherein the mass spectrometry comprises selected reaction monitoring (SRM) using a triple quadrupole mass spectrometer.
(Item 20A)
The method according to any one of items 15A to 19A, further comprising the step of detecting, identifying or quantifying the fatty acid side chains and/or phosphate groups of the lysophosphatidylinositol phosphate using liquid column chromatography or ion mobility separation.
(Item 21A)
The method according to any one of Items 15A to 20A, further comprising identifying the position of the phosphate group.
(Item 22A)
The method according to any one of Items 15A to 21A, wherein in the mass spectrometry, acylglycerol is selected as a product ion (fragment ion) to detect, identify or quantify the fatty acid side chain and/or phosphate group.
(Item 23A)
A method for diagnosing cancer by detecting, identifying or quantifying the cancer according to any one of items 15A to 22A.
(Item 24A)
A method for measuring, detecting or identifying phosphatidylinositol phosphate and/or lysophosphatidylinositol phosphate in a sample, the method comprising the steps of A) subjecting the sample to mass spectrometry (MS); and B) identifying the positions of the phosphate groups of the phosphatidylinositol phosphate and/or lysophosphatidylinositol phosphate by the elution position of a peak in the MS.
(Item 25A)
The method according to Item 24A, wherein in the step A), the sample is further subjected to chromatography.
(Item 26A)
the mass spectrometry is liquid column chromatography-mass spectrometry (LC-MS);
The method according to Item 24A or 25A, wherein the identifying step B) is a step of identifying the position of the phosphate group of the phosphatidylinositol phosphate and/or lysophosphatidylinositol phosphate based on the elution position of the peak in the LC-MS.
(Item 27A)
The method according to any one of Items 24A to 26A, wherein the total molecular weight of the acyl groups contained in the phosphatidylinositol phosphate is also identified in the identifying step.
(Item 28A)
The method according to any one of Items 24A to 27A, wherein the type of acyl group contained in the phosphatidylinositol phosphate and/or lysophosphatidylinositol phosphate is also identified in the identifying step.
(Item 29A)
The method according to any one of items 24A to 28A, wherein the positions of the phosphate groups of the phosphatidylinositol phosphate and the lysophosphatidylinositol phosphate are identified in the same run.
(Item 30A)
The method according to any one of items 24A to 29A, characterized in that the measuring, detecting or identifying is performed by distinguishing phosphatidylinositol phosphates and/or lysophosphatidylinositol phosphates from other inositol-containing phospholipids.
(Item 31A)
The method according to any one of Items 24A to 30A, wherein the sample is prepared under conditions that do not cause decomposition of phosphatidylinositol phosphates and/or lysophosphatidylinositol phosphates.
(Item 32A)
The method according to Item 31A, wherein the conditions include not performing a deacylation treatment.
(Item 33A)
The method according to any one of Items 26A to 32A, wherein the liquid column chromatography is selected from the group consisting of a chiral column, a reversed-phase column, and column chromatography that separates hydrophilic and hydrophobic groups.
(Item 34A)
The method according to any one of items 24A to 33A, wherein the liquid column chromatography is combined with ion mobility separation.
(Item 35A)
The method according to any one of items 24A to 34A, further comprising a step of quantifying the phosphatidylinositol phosphate and/or lysophosphatidylinositol phosphate.
(Item 36A)
The method of any one of items 24A to 35A, wherein the sample is an intact sample.
(Item 37A)
The method according to any one of items 24A to 36A, wherein the identification is performed without labeling the sample.
(Item 38A)
The mass spectrometry is ion mobility separation-mass spectrometry (IMS-MS),
The step B) of identifying is a step of identifying the positions of phosphate groups of the phosphatidylinositol phosphate and/or lysophosphatidylinositol phosphate based on the elution positions of the peaks in the IMS-MS.
The method according to any one of Items 24A to 37A.
(Item 39A)
The method according to item 38A, comprising any one or more of the features of items 25A to 33A and 35A to 37A.
(Item 40A)
An apparatus for measuring, detecting or identifying the positions of acyl and phosphate groups of phosphatidylinositol phosphate and/or lysophosphatidylinositol phosphate in a sample, comprising a mass spectrometry (MS) and an application section for applying the sample to the MS under conditions in which phosphatidylinositol phosphate is not decomposed.
(Item 41A)
The apparatus according to item 40A, further comprising an apparatus for chromatography.
(Item 42A)
The apparatus according to item 40A or 41A, wherein the mass spectrometry is liquid column chromatography-mass spectrometry (LC-MS), and the application unit is an application unit that applies the sample to the LC-MS under conditions in which phosphatidylinositol phosphate is not decomposed.
(Item 43A)
The apparatus according to Item 42A, wherein the liquid column chromatography is selected from the group consisting of all column chromatography that separates hydrophilic groups and hydrophobic groups, such as chiral columns and reverse phase columns.
(Item 44A)
The device of any one of claims 40A to 43A, further comprising a means for detecting or quantifying said phosphatidylinositol phosphates and/or lysophosphatidylinositol phosphates.
(Item 45A)
The apparatus according to any one of Items 40A to 44A, wherein the mass spectrometry is ion mobility separation-mass spectrometry (IMS-MS), and the application unit is an application unit that applies the sample to the IMS-MS under conditions in which phosphatidylinositol phosphate is not decomposed.
(Item 46A)
A method for separating or purifying phosphatidylinositol phosphates and/or lysophosphatidylinositol phosphates in a sample according to the position of the phosphate group while retaining the acyl group, the method comprising the steps of: A) applying the sample to liquid column chromatography-mass spectrometry (LC-MS) or ion mobility separation-mass spectrometry (IMS-MS); and B) collecting phosphatidylinositol phosphates and/or lysophosphatidylinositol phosphates having a phosphate group at a position of interest specified by the elution position of a peak from the LC or IMS eluate in the LC-MS or IMS-MS.
(Item 47A)
1. A method for producing, from a mixture in which phosphatidylinositol phosphates and/or lysophosphatidylinositol phosphates are present at two or more phosphate group positions, a sample containing phosphatidylinositol phosphates and/or lysophosphatidylinositol phosphates having fewer types of phosphate group positions than the number of types of phosphate group positions of the phosphatidylinositol phosphates and/or lysophosphatidylinositol phosphates contained in the mixture, comprising:
A) subjecting the sample to liquid column chromatography-mass spectrometry (LC-MS) or ion mobility separation-mass spectrometry (IMS-MS); and B) collecting, from the LC or IMS eluate in the LC-MS or IMS-MS, a fraction containing phosphatidylinositol phosphate and/or lysophosphatidylinositol phosphate having a phosphate group at a position of interest specified by the elution position of a peak.
(Item 48A)
The method according to Item 47A, wherein the number of types of phosphate group positions of phosphatidylinositol phosphates and/or lysophosphatidylinositol phosphates contained in the mixture is one type.

Furthermore, the present invention provides the following:
(1B) A method for measuring, detecting or identifying phosphatidylinositol phosphate in a sample, the method comprising the steps of A) subjecting the sample to liquid column chromatography-mass spectrometry (LC-MS); and B) identifying the position of the phosphate group of the phosphatidylinositol phosphate by the elution position of a peak in the LC-MS.
(2B) The method according to item 1B, wherein the presence or absence of an acyl group contained in the phosphatidylinositol phosphate is also identified in the identifying step.
(3B) The method according to item 1B or 2B, wherein the total molecular weight of the acyl groups contained in the phosphatidylinositol phosphate is also identified in the identifying step.
(4B) The method according to any one of items 1B to 3B, wherein the type of acyl group contained in the phosphatidylinositol phosphate is also identified in the identifying step.
(5B) The method according to any one of items 1B to 4B, wherein the measurement, detection or identification is performed by distinguishing phosphatidylinositol phosphate from other inositol-containing phospholipids.
(6B) The method according to any one of items 1B to 5B, wherein the sample is prepared under conditions in which phosphatidylinositol phosphate is not decomposed.
(7B) The method according to Item 6B, wherein the conditions include not performing a deacylation treatment.
(8B) The method according to any one of Items 1B to 7B, wherein the liquid column chromatography is selected from the group consisting of chiral columns, reverse phase columns, and column chromatography that separates hydrophilic groups and hydrophobic groups.
(9B) The method according to any one of items 1B to 8B, wherein the liquid column chromatography is combined with ion mobility separation.
(10B) The method according to any one of items 1B to 9B, further comprising a step of quantifying the phosphatidylinositol phosphate.
(11B) The method according to any one of items 1B to 10B, wherein the sample is an intact sample.
(12B) The method according to any one of items 1B to 11B, wherein the identification is performed without labeling the sample.
(13B) A method for measuring, detecting or identifying phosphatidylinositol phosphate in a sample, the method comprising the steps of A) subjecting the sample to ion mobility separation-mass spectrometry (IMS-MS); and B) identifying the position of the phosphate group of the phosphatidylinositol phosphate by the elution position of a peak in the IMS-MS.
(14B) The method according to item 13B, comprising any one or more of the features of items 2B to 8B and 10B to 12B.
(15B) An apparatus for measuring, detecting or identifying the positions of acyl groups and phosphate groups of phosphatidylinositol phosphate in a sample, comprising a liquid column chromatography-mass spectrometry (LC-MS) and an application section for applying the sample to the LC-MS under conditions that do not cause decomposition of phosphatidylinositol phosphate.
(16B) The apparatus according to item 15B, wherein the liquid column chromatography is selected from the group consisting of all column chromatography that separates hydrophilic groups and hydrophobic groups, such as chiral columns and reverse phase columns.
(17B) The device according to item 15B or 16B, further comprising a means for detecting or quantifying the phosphatidylinositol phosphate.
(17B2) An apparatus according to any one of items 15B to 17B, comprising any one or more features of items 2B to 12B.
(18B) An apparatus for measuring, detecting or identifying the positions of acyl groups and phosphate groups of phosphatidylinositol phosphate in a sample, comprising an ion mobility separation-mass spectrometry (IMS-MS) and an application section for applying the sample to the IMS-MS under conditions in which phosphatidylinositol phosphate is not decomposed.
(18B2) The device according to item 18B, comprising any one or more of the features of items 2B to 12B.
(19B) A method for separating or purifying phosphatidylinositol phosphates in a sample according to the position of the phosphate group while retaining the acyl group, the method comprising the steps of A) subjecting the sample to liquid column chromatography-mass spectrometry (LC-MS) or ion mobility separation-mass spectrometry (IMS-MS); and B) collecting, from the LC-MS or IMS-MS eluate, phosphatidylinositol phosphates having a phosphate group at a target position identified by the elution position of a peak.
(19B2) The method according to item 19B having any one or more features of items 2B to 12B.
(20B) A method for producing, from a mixture in which phosphatidylinositol phosphates have two or more phosphate group positions, a sample containing phosphatidylinositol phosphates having phosphate groups at positions with fewer types of phosphate group positions than the number of types of phosphate group positions of phosphatidylinositol phosphates contained in the mixture, comprising the steps of:
A) subjecting the sample to liquid column chromatography-mass spectrometry (LC-MS) or ion mobility separation-mass spectrometry (IMS-MS); and B) collecting, from the LC-MS or IMS-MS eluate, a fraction containing a phosphatidylinositol phosphate having a phosphate group at a position of interest identified by the elution position of a peak.
(21B) The method according to Item 20B, wherein the number of types of positions of phosphate groups of phosphatidylinositol phosphates contained in the mixture is one type.
(21B2) The method according to item 20B or 21B, comprising any one or more features of items 2B to 12B.

Previous analytical methods were unable to separate phosphorylation positional isomers and measured the sum of the three isomers, but we have developed a method that can separate and measure isomers by using mass spectrometry (MS) in combination with chromatography or ion mobility separation (IMS) as necessary. In one embodiment, the present invention relates to an analytical method that can separate and measure eight types of PIPs and LPIPs isomers in an intact state by using a chiral column. An exemplary analysis uses MRM/SRM with a triple quadrapole mass spectrometer. Q1 is set as an intact precursor ion, and Q3 is set as a product ion of DAG or MAG from which inositol phosphate has been released.

The present invention also provides the following:
(1C) A compound which is lysophosphatidylinositol monophosphate, lysophosphatidylinositol diphosphate or lysophosphatidylinositol triphosphate.
(2C) The compound according to item 1C, which is 1-acyl-lysophosphatidylinositol monophosphate, 2-acyl-lysophosphatidylinositol monophosphate, 1-acyl-lysophosphatidylinositol diphosphate, 2-acyl-lysophosphatidylinositol diphosphate, 1-acyl-lysophosphatidylinositol triphosphate, or 2-acyl-lysophosphatidylinositol triphosphate.
(3C) 1-acyl-lysophosphatidylinositol-3-monophosphate, 1-acyl-lysophosphatidylinositol-4-monophosphate, 1-acyl-lysophosphatidylinositol-5-monophosphate, 2-acyl-lysophosphatidylinositol-3-monophosphate, 2-acyl-lysophosphatidylinositol-4-monophosphate, 2-acyl-lysophosphatidylinositol-5-monophosphate, 1-acyl-lysophosphatidylinositol-3.4-diphosphate, 1-acyl-lysophosphatidylinositol The compound according to item 1C or 2C is 1-acyl-lysophosphatidylinositol-3.5-diphosphate, 1-acyl-lysophosphatidylinositol-4.5-diphosphate, 2-acyl-lysophosphatidylinositol-3,4-diphosphate, 2-acyl-lysophosphatidylinositol-3,5-diphosphate, 2-acyl-lysophosphatidylinositol-4,5-diphosphate, 1-acyl-lysophosphatidylinositol-3,4,5-triphosphate, or 2-acyl-lysophosphatidylinositol-3,4,5-triphosphate.
(4C) The compound according to any one of items 1C to 3C, which is 2-acyl-lysophosphatidylinositol monophosphate, 2-acyl-lysophosphatidylinositol diphosphate, 1-acyl-lysophosphatidylinositol triphosphate or 2-acyl-lysophosphatidylinositol triphosphate.
(5C) 1-butanyl-lysophosphatidylinositol 4-monophosphate;
1-Hexanyl-lysophosphatidylinositol 4-monophosphate;
1-octanyl-lysophosphatidylinositol 4-monophosphate;
1-Hexadecanyl-lysophosphatidylinositol 4-monophosphate;
1-9Z-hexadecenyl-lysophosphatidylinositol 4-monophosphate;
1-heptadecanyl-lysophosphatidylinositol 4-monophosphate;
1-Octadecanyl-lysophosphatidylinositol 4-monophosphate;
1-9Z-octadecenyl-lysophosphatidylinositol 4-monophosphate;
1-9Z,12Z-octadecanedienyl-lysophosphatidylinositol 4-monophosphate;
1-8Z,11Z,14Z-icosatrienyl-lysophosphatidylinositol 4-monophosphate;
1-5Z,8Z,11Z,14Z-icosatetraenyl-lysophosphatidylinositol 4-monophosphate;
1-7Z,10Z,13Z,16Z-docosatetraenyl-lysophosphatidylinositol 4-monophosphate;
1-7Z,10Z,13Z,16Z,19Z-docosapentaenyl-lysophosphatidylinositol 4-monophosphate;
1-4Z,7Z,10Z,13Z,16Z,19Z-docosahexaenyl-lysophosphatidylinositol 4-monophosphate;
2-butanyl-lysophosphatidylinositol 4-monophosphate;
2-Hexanyl-lysophosphatidylinositol 4-monophosphate;
2-octanyl-lysophosphatidylinositol 4-monophosphate;
2-Hexadecanyl-lysophosphatidylinositol 4-monophosphate;
2-9Z-hexadecenyl-lysophosphatidylinositol 4-monophosphate;
2-heptadecanyl-lysophosphatidylinositol 4-monophosphate;
2-octadecanyl-lysophosphatidylinositol 4-monophosphate;
2-9Z-octadecenyl-lysophosphatidylinositol 4-monophosphate;
2-9Z,12Z-octadecanedienyl-lysophosphatidylinositol 4-monophosphate;
2-8Z,11Z,14Z-icosatrienyl-lysophosphatidylinositol 4-monophosphate;
2-5Z,8Z,11Z,14Z-icosatetraenyl-lysophosphatidylinositol 4-monophosphate;
2-7Z,10Z,13Z,16Z-docosatetraenyl-lysophosphatidylinositol 4-monophosphate;
2-7Z,10Z,13Z,16Z,19Z-docosapentaenyl-lysophosphatidylinositol 4-monophosphate;
2-4Z,7Z,10Z,13Z,16Z,19Z-docosahexaenyl-lysophosphatidylinositol 4-monophosphate;
1-butanyl-lysophosphatidylinositol 4,5-bisphosphate;
1-Hexanyl-lysophosphatidylinositol 4,5-bisphosphate;
1-octanyl-lysophosphatidylinositol 4,5-bisphosphate;
1-Hexadecanyl-lysophosphatidylinositol 4,5-bisphosphate;
1-9Z-hexadecenyl-lysophosphatidylinositol 4,5-bisphosphate;
1-heptadecanyl-lysophosphatidylinositol 4,5-bisphosphate;
1-Octadecanyl-lysophosphatidylinositol 4,5-bisphosphate;
1-9Z-octadecenyl-lysophosphatidylinositol 4,5-bisphosphate;
1-9Z,12Z-octadecanedienyl-lysophosphatidylinositol 4,5-bisphosphate;
1-6Z,9Z,12Z-octadecanedienyl-lysophosphatidylinositol 4,5-bisphosphate;
1-8Z,11Z,14Z-icosatrienyl-lysophosphatidylinositol 4,5-bisphosphate;
1-5Z,8Z,11Z,14Z-icosatetraenyl-lysophosphatidylinositol 4,5-bisphosphate;
2-butanyl-lysophosphatidylinositol 4,5-bisphosphate;
2-Hexanyl-lysophosphatidylinositol 4,5-bisphosphate;
2-octanyl-lysophosphatidylinositol 4,5-bisphosphate;
2-Hexadecanyl-lysophosphatidylinositol 4,5-bisphosphate;
2-9Z-hexadecenyl-lysophosphatidylinositol 4,5-bisphosphate;
2-heptadecanyl-lysophosphatidylinositol 4,5-bisphosphate;
2-octadecanyl-lysophosphatidylinositol 4,5-bisphosphate;
2-9Z-octadecenyl-lysophosphatidylinositol 4,5-bisphosphate;
2-9Z,12Z-octadecanedienyl-lysophosphatidylinositol 4,5-bisphosphate;
2-6Z,9Z,12Z-octadecanedienyl-lysophosphatidylinositol 4,5-bisphosphate;
2-8Z,11Z,14Z-icosatrienyl-lysophosphatidylinositol 4,5-bisphosphate;
2-5Z,8Z,11Z,14Z-icosatetraenyl-lysophosphatidylinositol 4,5-bisphosphate;
1-butanyl-lysophosphatidylinositol 3,4,5-triphosphate;
1-octanyl-lysophosphatidylinositol 3,4,5-triphosphate;
1-Hexadecanyl-lysophosphatidylinositol 3,4,5-triphosphate;
1-9Z-hexadecenyl-lysophosphatidylinositol 3,4,5-triphosphate;
1-heptadecanyl-lysophosphatidylinositol 3,4,5-triphosphate;
1-Octadecanyl-lysophosphatidylinositol 3,4,5-triphosphate;
1-9Z-octadecenyl-lysophosphatidylinositol 3,4,5-triphosphate;
1-5Z,8Z,11Z,14Z-icosatetraenyl-lysophosphatidylinositol 3,4,5-triphosphate;
1-4Z,7Z,10Z,13Z,16Z,19Z-docosahexaenyl-lysophosphatidylinositol 3,4,5-triphosphate;
2-butanyl-lysophosphatidylinositol 3,4,5-triphosphate;
2-octanyl-lysophosphatidylinositol 3,4,5-triphosphate;
2-Hexadecanyl-lysophosphatidylinositol 3,4,5-triphosphate;
2-9Z-hexadecenyl-lysophosphatidylinositol 3,4,5-triphosphate;
2-heptadecanyl-lysophosphatidylinositol 3,4,5-triphosphate;
2-octadecanyl-lysophosphatidylinositol 3,4,5-triphosphate;
2-9Z-octadecenyl-lysophosphatidylinositol 3,4,5-triphosphate;
2-5Z,8Z,11Z,14Z-icosatetraenyl-lysophosphatidylinositol 3,4,5-triphosphate; and 2-4Z,7Z,10Z,13Z,16Z,19Z-docosahexaenyl-lysophosphatidylinositol 3,4,5-triphosphate,
The compound according to any one of items 1C to 4C.
(6C) A composition comprising the compound according to any one of items 1C to 5C for use as a cancer marker.
(7C) The composition of item 6C, wherein the cancer comprises prostate cancer.
(8C) A method for producing the compound according to any one of Items 1C to 5C or the composition according to Items 6C or 7C, comprising a step of deacylating phosphatidylinositol monophosphate, phosphatidylinositol diphosphate or phosphatidylinositol triphosphate by contacting it with an alkylamine.
(9C) The method according to item 8C, wherein the alkylamine is methylamine.
(10C) The method according to item 8C or 9C, wherein the deacylation is carried out under milder conditions than the conditions for dediacylation.
(11C) The method according to Item 10C, wherein the mild conditions are achieved by shortening the reaction time, reducing the concentration of the alkylamine, reducing the reaction temperature, or a combination thereof among the dediacylation conditions.
(12C) The method according to item 10C or 11C, wherein the mild conditions are: use of methylamine as the alkylamine; a reaction time of 10 minutes or less; a methylamine concentration of about 11% or less; and a reaction temperature of 53° C. or less; or a combination of the reaction time, the concentration, and the reaction temperature.
(13C) A method for producing lysophosphatidylinositol monophosphate, lysophosphatidylinositol diphosphate or lysophosphatidylinositol triphosphate having a desired acyl group at a desired position, comprising: (A) providing phosphatidylinositol monophosphate, phosphatidylinositol diphosphate or phosphatidylinositol triphosphate having acyl groups of different masses at the 1-position and the 2-position, wherein the acyl group having the desired mass is present at a desired position selected from the 1-position and the 2-position; (B) deacylating the phosphatidylinositol monophosphate, phosphatidylinositol diphosphate or phosphatidylinositol triphosphate by contacting it with an alkylamine; and (C) as necessary, isolating lysophosphatidylinositol monophosphate, lysophosphatidylinositol diphosphate or lysophosphatidylinositol triphosphate having an acyl group of the desired mass.
(14C) A method for detecting, identifying or quantifying lysophosphatidylinositol phosphate, comprising the steps of: (A) concentrating phospholipids by contacting a sample with an anion exchange resin; (B) protecting phosphate groups in the acidic phospholipids with protecting groups; and (C) detecting, identifying or quantifying lysophosphatidylinositol phosphate in the phospholipids by mass spectrometry.
(15C) The method according to item 14C, wherein the protecting group comprises an alkyl group.
(16C) The method according to item 14C or 15C, wherein the protecting group comprises a methyl group.
(17C) The method according to any one of Items 14C to 16C, wherein the mass spectrometry method includes selected reaction monitoring (SRM) using a triple quadrupole mass spectrometer.
(18C) The method according to any one of items 14C to 17C, further comprising detecting, identifying or quantifying the fatty acid side chain and/or phosphate group of the lysophosphatidylinositol phosphate using reverse phase column chromatography.
(19C) The method according to Item 18C, wherein diacylglycerol is selected as a product ion (fragment ion) in the reverse phase column chromatography to detect, identify or quantify the fatty acid side chain and/or phosphate group.
(20C) A method for diagnosing cancer by detecting, identifying or quantifying the cancer according to any one of items 14C to 19C.

In the present invention, it is intended that one or more of the above features may be provided in combinations other than those explicitly stated. Still further embodiments and advantages of the present invention will be recognized by those skilled in the art upon reading and understanding the following detailed description, if necessary.

By providing new lipids, the present invention can elucidate the vital functions of these lipids and promote medical applications. It can also present new targets for drug discovery. Furthermore, by providing new measurement methods, the present invention can provide new diagnostic and detection methods.

Furthermore, by using the present invention, phosphoinositides and/or lysophosphoinositides can be separated at the position of the phosphate group in an intact state and measured, detected or identified, allowing detailed analysis within the living body to be performed. Such information is useful for biological information, pharmacological information, drug development, treatment, diagnosis, etc.

FIG. 1A is a schematic diagram illustrating the principle of the "measurement and detection method by SRM using a triple quadrupole mass spectrometer." 1B shows the results of an experiment to examine the conditions for synthesizing the compound of the present invention. This is an example showing that LPIP1 is not produced under conventional deacylation reaction conditions, but can be obtained by shortening the reaction time (upper row) or decreasing the methylamine concentration (lower row). The vertical axis shows the yield (%) of the reaction product, and the horizontal axis shows the time (min). 1C shows the results of the investigation of reaction temperature. 17:0/20:4 PI4P was used as the raw material. The products were 17:0 LPIP1 and 20:4 LPIP1. The vertical axis shows the yield (%) of the LPIP1 product. Figures 2A to 2G show that seven types of LPIPs with different phosphorylation patterns can be synthesized by deacylation under the mild conditions shown in Figure 1. In each case, the production of LPIPs with 17:0 and 20:4 acyl groups is shown as an example to show that LPIPs with different acyl groups can be synthesized. Figure 2A shows the identification data of the synthesis of the compound of the present invention. Raw material: 17:0/20:4 PI3P, target product: 17:0 LPI3P (top): ms1 = 754.390, ms2 = 327.290; 20:4 LPI3P (bottom): ms1 = 788.370, ms2 = 361.270. The vertical axis indicates the relative abundance, and the horizontal axis indicates time (min). Figure 2B shows the identification data of the synthesis of the compound of the present invention. Raw material: 17:0/20:4 PI4P, target product: 17:0 LPI4P (top): ms1 = 754.390, ms2 = 327.290; 20:4 LPI4P (bottom): ms1 = 788.370, ms2 = 361.270. The vertical axis shows the relative abundance, and the horizontal axis shows time (min). Figure 2C shows the identification data of the synthesis of the compound of the present invention. Raw material: 17:0/20:4 PI5P, target product: 17:0 LPI5P (top): ms1 = 754.390, ms2 = 327.290; 20:4 LPI5P (bottom): ms1 = 788.370, ms2 = 361.270. The vertical axis shows the relative abundance, and the horizontal axis shows time (min). Figure 2D shows the identification data of the synthesis of the compound of the present invention. Raw material: 17:0/20:4 PI(3,4)P2, target product: 17:0 LPI(3,4)P2 (top): ms1 = 862.390, ms2 = 327.290; 20:4 LPI(3,4)P2 (bottom): ms1 = 896.370, ms2 = 361.270. The vertical axis shows the relative abundance, and the horizontal axis shows time (min). Figure 2E shows the identification data of the synthesis of the compound of the present invention. Raw material: 17:0/20:4 PI(3,5)P2, target product: 17:0 LPI(3,5)P2 (top): ms1 = 862.390, ms2 = 327.290; 20:4 LPI(3,5)P2 (bottom): ms1 = 896.370, ms2 = 361.270. The vertical axis shows the relative abundance, and the horizontal axis shows time (min). Figure 2F shows the identification data of the synthesis of the compound of the present invention. Raw material: 17:0/20:4 PI(4,5)P2, target product: L17:0 PI(4,5)P2 (top): ms1 = 862.390, ms2 = 327.290; 20:4 LPI(4,5)P2 (bottom): ms1 = 896.370, ms2 = 361.270. The vertical axis shows the relative abundance, and the horizontal axis shows time (min). Figure 2G shows the identification data of the synthesis of the compound of the present invention. Raw material: 17:0/20:4 PI(3,4,5)P3, target product: 17:0 LPI(3,4,5)P3 (top): ms1 = 970.390, ms2 = 327.290; 20:4 LPI(3,4,5)P3 (bottom): ms1 = 1004.370, ms2 = 361.270. The vertical axis shows the relative abundance, and the horizontal axis shows time (min). 3A to 3F show the results of the identification of the structures of the present invention (confirmation of the structures of LPIP1, LPIP2, and LPIP3) by measuring the exact mass using a Thermo Fisher Scientific ^™ Q Exactive ^™ plus (hybrid quadrupole-orbitrap mass spectrometer). FIG. 3A shows the results of accurate mass analysis of LPI(4)P and fragments derived from LPI(4)P for two types of LPI(4)P generated by mild deacylation reaction of 37:4 PI(4)P (sn-1 17:0, sn-2 20:4 PI(4)P) (see FIG. 1). The vertical axis shows the relative abundance, and the horizontal axis shows time (min). FIG. 3B shows the structural formulae of 37:4 PI(4)P, the product after the deacylation reaction, and its fragments. Figure 3C shows the results of accurate mass spectrometry of LPI(4,5)P2 and fragments derived from LPI(4,5)P2 for two types of LPI(4,5)P2 generated by mild deacylation of 37:4 PI(4,5)P2 (sn-1 17:0, sn-2 20:4 PI(4,5)P2). The vertical axis indicates relative abundance, and the horizontal axis indicates time (min). FIG. 3D shows the structural formulae of 37:4 PI(4,5)P2, the product after deacylation reaction, and its fragments. Figure 3E shows the results of accurate mass spectrometry of LPI(3,4,5)P3 and fragments derived from LPI(3,4,5)P3 for two types of LPI(3,4,5)P3 generated by mild deacylation of 37:4 PI(3,4,5)P3 (sn-1 17:0, sn-2 20:4 PI(3,4,5)P3). The vertical axis indicates relative abundance, and the horizontal axis indicates time (min). FIG. 3F shows the structural formulae of 37:4 PI(3,4,5)P3, the product after the deacylation reaction, and its fragments. Figure 4A shows an overview of the spin system of an inositol phospholipid. The solid box indicates a group of ^1H nuclei connected by continuous ^3J couplings, and the dashed box indicates a group of ^1H and ^31P nuclei that are ^3J coupled. FIG. 4B shows the chemical structure observed from the NMR spectrum of Brain PI4P (Avanti Polar Lipids, Alabama, USA). The structural formula of 1-stearoyl-2-arachidonyl-sn-glycero-3-phospho-(1'-myo-inositol-4'-monophosphate) and the chemical structure observed from the signals in the ^1H -1D and TOCSY spectra are shown. Signals of fatty acids, glycerol and inositol were observed in full. The _CH2 of C1 was magnetically nonequivalent. The _aCH2 , _bCH2 and _gCH2 of arachidonic acid were observed separately. FIG. 4C shows the chemical structure of Sample 1 (16:0 LPI4P) of Example 1 observed from the ¹ H-1D and TOCSY spectra. The structural formula of 16:0 LPI4P and the chemical structure observed from the ¹ H-1D and TOCSY spectra are shown. Signals of fatty acids and C1 and C3 of the glycerol skeleton were observed. A weak signal thought to be an inositol ring was observed in the region around 4 ppm. No signal of polyunsaturated fatty acids was observed. FIG. 4D shows the chemical structure of Sample 2 (17:0 LPI(4,5)P2) in Example 1, observed from the ¹ H-1D and TOCSY spectra. The structural formula of 17:0 LPI(4,5)P2 and the chemical structure observed from the signals in the ¹ H-1D and TOCSY spectra are shown. Signals of fatty acid and C1 and C3 of the glycerol backbone were observed. Minor signals of C1, C3, aCH ₂ (and bCH ₂ ) were observed. A weak signal thought to be an inositol ring was observed in the region around 4 ppm. FIG. 4E shows the chemical structure of Sample 3 (18:0 LPI(4,5)P2) in Example 1, observed from the ¹ H-1D and TOCSY spectrum. The structural formula of 18:0 LPI(4,5)P2 and the chemical structure observed from the signals in the ¹ H-1D and TOCSY spectrum are shown. Signals of fatty acid and C1 and C3 of the glycerol backbone were observed. Minor signals of C1, C3, aCH ₂ (and bCH ₂ ) were observed, which were weaker than those of Samples 1 and 2. A signal thought to be an inositol ring was weakly observed in the region around 4 ppm. FIG. 4F shows the chemical structure of Sample 5 (17:0/20:4 PI(4,5)P2) in Example 1, as observed from the ¹ H-1D and TOCSY spectrum. The structural formula of 17:0/20:4 PI(4,5)P2 and the chemical structure observed from the signals in the ¹ H-1D and TOCSY spectrum are shown. Signals of C1, C2, and C3 of the fatty acid and glycerol backbone were observed. aCH ₂ , bCH ₂ , and gCH ₂ of arachidonic acid were observed separately. Signals of polyunsaturated fatty acids (-CH=CH-CH ₂ -CH=CH-, -CH=CH-) were hardly observed. 5A to 5D show data on the presence of the compound of the present invention in biological samples (cultured cancer cell lines). Fig. 5A shows the presence of LPIPs in HEK293T cells. The vertical axis shows the amount (pmol) of the compound present relative to 1 nmol of phosphatidylserine (PS), and the horizontal axis shows the molecular species. 5B shows the presence of LPIPs in Jurkat cells. The vertical axis represents the amount (pmol) of a compound present relative to 1 nmol of phosphatidylserine (PS), and the horizontal axis represents the molecular species. 5C shows the results of accurate mass measurement of LPIP3 and its fragments present in HEK293T cells. The vertical axis indicates the relative abundance, and the horizontal axis indicates m/z (mass/charge number). Fig. 5D shows the agreement of the elution times of LPIP3 and its fragments present in HEK293T cells. The vertical axis indicates the relative abundance, and the horizontal axis indicates time (min). Figures 6A to 6D show data on the presence of the compounds of the present invention in biological samples (mouse, human). Figure 6A shows the presence of LPIPs in mouse heart, large intestine, liver, brain (whole brain), and spleen. The vertical axis shows the amount (pmol) of compound present per nmol of phosphatidylserine (PS), and the horizontal axis shows the type of cell. 6B shows the presence of LPIPs in mouse serum. The vertical axis represents the amount (fmol) of compound present per 1 μL of serum, and the horizontal axis represents molecular species. 6C shows the presence of LPIPs in mouse plasma. The vertical axis represents the amount (fmol) of compound present per 1 μL of plasma, and the horizontal axis represents molecular species. 6D shows the presence of LPIPs in human serum. The vertical axis represents the amount (fmol) of compound present per 1 μL of serum, and the horizontal axis represents molecular species. FIG. 7A shows mouse prostate weight (vertical axis indicates weight (mg) and horizontal axis indicates tissue type) in the upper row, and HE-stained images of the prostate in the lower row, demonstrating that mice lacking Pten develop prostate cancer. 7B shows that LPIPs levels in the prostate increase with the onset of prostate cancer. The vertical axis represents the LPIPs level (A.U. (arbitrary unit)) relative to 1 nmol of phosphatidylserine (PS), and Ctrl on the horizontal axis represents normal mice, and PTEN KO represents mice in which the tumor suppressor gene Pten is specifically deleted in the prostate. 8 shows that LPIP3 is produced in bone marrow cells following stimulation with complement component 5a, an inflammatory substance. The vertical axis indicates the amount (pmol) of LPIP3 present in ¹⁰⁶ cells, and "-" on the horizontal axis indicates that stimulation with complement component 5a has not been performed, and "+" indicates that stimulation with complement component 5a has been performed. 9A and 9B are diagrams showing the separation of 17:0/20:4 phosphoinositides by a chiral column. Each graph is a chromatogram obtained by measuring a phosphoinositide sample of the indicated phosphate group by a mass spectrometer, with the horizontal axis representing the retention time (min) and the vertical axis representing the relative signal intensity when the signal intensity (cps) at the peak top is taken as 100%. The left column of FIG. 9A shows PI, the center column shows, from the top, PI(3)P, PI(4)P, and PI(5)P, respectively, and the right column shows their mixture (PIP1mix). The second column from the right shows PI(3,5)P, PI(3,4)P2PI(4,5)P2, and their mixture, respectively. The left column of FIG. 9B shows, from the top, PI(3,5)P2, PI(3,4)P2, and PI(4,5)P2, respectively, the center shows a mixture thereof (PIP2mix), and the right column shows PI(3,4,5)P3. 9A and 9B are diagrams showing the separation of 17:0/20:4 phosphoinositides by a chiral column. Each graph is a chromatogram obtained by measuring a phosphoinositide sample of the indicated phosphate group by a mass spectrometer, with the horizontal axis representing the retention time (min) and the vertical axis representing the relative signal intensity when the signal intensity (cps) at the peak top is taken as 100%. The left column of FIG. 9A shows PI, the center column shows, from the top, PI(3)P, PI(4)P, and PI(5)P, respectively, and the right column shows their mixture (PIP1mix). The second column from the right shows PI(3,5)P, PI(3,4)P2PI(4,5)P2, and their mixture, respectively. The left column of FIG. 9B shows, from the top, PI(3,5)P2, PI(3,4)P2, and PI(4,5)P2, respectively, the center shows a mixture thereof (PIP2mix), and the right column shows PI(3,4,5)P3. FIG. 10 shows a calibration curve created from the signal intensity (peak area) of seven different amounts (0.02, 0.05, 0.1, 0.2, 5, 10, 50 pmol) of the indicated phosphate group of 17:0/20:4 phosphoinositides when measured by a mass spectrometer (four repeated measurements at each injection amount). The average of the four repeated measurements is represented by black dots, and the standard deviation is represented by error bars. The upper row shows PI, PI(3)P, and PI(4)P from the left, the middle row shows PI(5)P, PI(3,5)P, and PI(3,4)P2 from the left, and the lower row shows PI(4,5)P2, PI(3,4,5), and P3 from the left. FIG. 11 shows the variation in signal intensity (cps) when 10 pmol of 17:0/20:4 phosphoinositides of the indicated phosphate groups were repeatedly measured 50 times by a mass spectrometer. The vertical axis represents signal intensity (cps), and the horizontal axis represents the number of injections. In the 10th injection, PI(3)P gave the strongest signal intensity, PI(4)P gave the second strongest signal intensity, PI(5)P gave the third strongest signal intensity, PI(3,4)P2 gave the fourth strongest signal intensity, PI(3,5)P2 gave the fifth strongest signal intensity, PI gave the sixth strongest signal intensity, PI(4,5)P2 gave the seventh strongest signal intensity, and PI(3,4,5)P3 gave the weakest signal intensity. FIG. 12 shows the separation of 17:0/20:4 phosphoinositides (PI(3,4)P2, PI(3,5)P2, PI(4,5)P2) by ion mobility. The horizontal axis represents the compensation voltage (COV, V), and the vertical axis represents the signal intensity (cps). The measurement results of each phosphoinositide are shown superimposed. Fig. 13 shows the change in phosphoinositide composition of cells when cells are treated _{with H2O2} , as measured by combining a chiral column and a mass spectrometer. In each graph, the horizontal axis represents retention time, and the vertical axis represents relative signal intensity when the _signal intensity (cps) at the peak top is taken as 100%. The graphs show the results at 0, 2, 5, and 15 minutes after _H2O2 treatment from the top, and the results for the masses corresponding to PI, PIP, PIP2, and PIP3 from the left. 14A-D show the change in phosphoinositide composition for each diacylglycerol of cells when cells are treated with H ₂ O ₂ and the total amount of each phosphoinositide as quantitative values calculated from the measurement results of a chiral column-mass spectrometer. In the graphs in the left column, the horizontal axis represents the type of diacylglycerol in the phosphoinositide, and the vertical axis represents the amount (pmol) of each phosphoinositide contained in 10 ⁶ cells calculated as the average of four measurements, together with the standard deviation. For each type of diacylglycerol, the results are shown from the left at 0 minutes, 2 minutes, 5 minutes, and 15 minutes after H ₂ O ₂ treatment. In the graphs in the right column, the vertical axis represents the total amount (pmol) of phosphoinositides having the same phosphate substitution contained in 10 ⁶ cells calculated as the average of four measurements, together with the standard deviation, and the horizontal axis represents the results from the left at 0 minutes, 2 minutes, 5 minutes, and 15 minutes after H ₂ O ₂ treatment. The results were obtained by repeating measurements four times, and the average of the repeated measurements is shown together with the standard deviation (error bars). When statistically processed using one-way analysis of variance and Tukey's multiple comparison test, ^*** indicates p<0.001, ^** indicates p<0.01, and ^* indicates p<0.05, respectively. 14A-D show the change in phosphoinositide composition for each diacylglycerol of cells when cells are treated with H ₂ O ₂ and the total amount of each phosphoinositide as quantitative values calculated from the measurement results of a chiral column-mass spectrometer. In the graphs in the left column, the horizontal axis represents the type of diacylglycerol in the phosphoinositide, and the vertical axis represents the amount (pmol) of each phosphoinositide contained in 10 ⁶ cells calculated as the average of four measurements, together with the standard deviation. For each type of diacylglycerol, the results are shown from the left at 0 minutes, 2 minutes, 5 minutes, and 15 minutes after H ₂ O ₂ treatment. In the graphs in the right column, the vertical axis represents the total amount (pmol) of phosphoinositides having the same phosphate substitution contained in 10 ⁶ cells calculated as the average of four measurements, together with the standard deviation, and the horizontal axis represents the results from the left at 0 minutes, 2 minutes, 5 minutes, and 15 minutes after H ₂ O ₂ treatment. The results were obtained by repeating measurements four times, and the average of the repeated measurements is shown together with the standard deviation (error bars). When statistically processed using one-way analysis of variance and Tukey's multiple comparison test, ^*** indicates p<0.001, ^** indicates p<0.01, and ^* indicates p<0.05, respectively. 14A-D show the change in phosphoinositide composition for each diacylglycerol of cells when cells are treated with H ₂ O ₂ and the total amount of each phosphoinositide as quantitative values calculated from the measurement results of a chiral column-mass spectrometer. In the graphs in the left column, the horizontal axis represents the type of diacylglycerol in the phosphoinositide, and the vertical axis represents the amount (pmol) of each phosphoinositide contained in 10 ⁶ cells calculated as the average of four measurements, together with the standard deviation. For each type of diacylglycerol, the results are shown from the left at 0 minutes, 2 minutes, 5 minutes, and 15 minutes after H ₂ O ₂ treatment. In the graphs in the right column, the vertical axis represents the total amount (pmol) of phosphoinositides having the same phosphate substitution contained in 10 ⁶ cells calculated as the average of four measurements, together with the standard deviation, and the horizontal axis represents the results from the left at 0 minutes, 2 minutes, 5 minutes, and 15 minutes after H ₂ O ₂ treatment. The results were obtained by repeating measurements four times, and the average of the repeated measurements is shown together with the standard deviation (error bars). When statistically processed using one-way analysis of variance and Tukey's multiple comparison test, ^*** indicates p<0.001, ^** indicates p<0.01, and ^* indicates p<0.05, respectively. 14A-D show the change in phosphoinositide composition for each diacylglycerol of cells when cells are treated with H ₂ O ₂ and the total amount of each phosphoinositide as quantitative values calculated from the measurement results of a chiral column-mass spectrometer. In the graphs in the left column, the horizontal axis represents the type of diacylglycerol in the phosphoinositide, and the vertical axis represents the amount (pmol) of each phosphoinositide contained in 10 ⁶ cells calculated as the average of four measurements, together with the standard deviation. For each type of diacylglycerol, the results are shown from the left at 0 minutes, 2 minutes, 5 minutes, and 15 minutes after H ₂ O ₂ treatment. In the graphs in the right column, the vertical axis represents the total amount (pmol) of phosphoinositides having the same phosphate substitution contained in 10 ⁶ cells calculated as the average of four measurements, together with the standard deviation, and the horizontal axis represents the results from the left at 0 minutes, 2 minutes, 5 minutes, and 15 minutes after H ₂ O ₂ treatment. The results were obtained by repeating measurements four times, and the average of the repeated measurements is shown together with the standard deviation (error bars). When statistically processed using one-way analysis of variance and Tukey's multiple comparison test, ^*** indicates p<0.001, ^** indicates p<0.01, and ^* indicates p<0.05, respectively. 15 shows the change in phosphoinositide composition in the thyroid gland (organ) of a mouse when the mouse was genetically modified, as a result of measurement using a chiral column in combination with a mass spectrometer. In each graph, the horizontal axis represents retention time, and the vertical axis represents relative signal intensity when the signal intensity (cps) at the peak top is taken as 100%. The graphs show the results for wild-type, INPP4B(-/-), PTEN(+/-), and INPP4B(-/-)PTEN(+/-) mice from the top, and the results for masses corresponding to PI, PIP, PIP2, and PIP3 from the left. 16A-C show the change in phosphoinositide composition for each diacylglycerol in the thyroid gland (organ) of a mouse when the mouse was genetically modified, and the total amount of each phosphoinositide, as a quantitative value calculated from the measurement results of a chiral column-mass spectrometer. In the graphs of FIG. 16A and B, the horizontal axis represents the type of diacylglycerol in the phosphoinositide, and the vertical axis represents the amount (pmol) of each phosphoinositide contained in 1 mg of tissue calculated as the average of three (wild type) and four (INPP4B(-/-), PTEN(+/-), and INPP4B(-/-)PTEN(+/-)) measurements, together with the standard deviation. For each type of diacylglycerol, the results for wild type, INPP4B(-/-), PTEN(+/-), and INPP4B(-/-)PTEN(+/-) mice are shown from the left. 16A-C show the change in phosphoinositide composition for each diacylglycerol in the thyroid gland (organ) of a mouse when the mouse was genetically modified, and the total amount of each phosphoinositide, as a quantitative value calculated from the measurement results of a chiral column-mass spectrometer. In the graphs of FIG. 16A and B, the horizontal axis represents the type of diacylglycerol in the phosphoinositide, and the vertical axis represents the amount (pmol) of each phosphoinositide contained in 1 mg of tissue calculated as the average of three (wild type) and four (INPP4B(-/-), PTEN(+/-), and INPP4B(-/-)PTEN(+/-)) measurements, together with the standard deviation. For each type of diacylglycerol, the results for wild type, INPP4B(-/-), PTEN(+/-), and INPP4B(-/-)PTEN(+/-) mice are shown from the left. Figures 16A-C show the change in phosphoinositide composition for each diacylglycerol in the thyroid gland (organ) of a mouse when the mouse was genetically modified, and the total amount of each phosphoinositide, as a quantitative value calculated from the measurement results of a chiral column-mass spectrometer. In the graph of Figure 16C, the vertical axis shows the total amount (pmol) of phosphoinositides having the same phosphate substitution contained in 1 mg of tissue calculated as the average of three (wild type) and four (INPP4B (-/-), PTEN (+/-), and INPP4B (-/-) PTEN (+/-)) measurements, together with the standard deviation, and the horizontal axis shows the results for wild type, INPP4B (-/-), PTEN (+/-), and INPP4B (-/-) PTEN (+/-) mice from the left. The results are from three (wild type) and four (INPP4B(-/-), PTEN(+/-), and INPP4B(-/-)PTEN(+/-)) repeated measurements, and the average of the repeated measurements is shown together with the standard deviation (error bars). When statistically processed using one-way analysis of variance and Tukey's multiple comparison test, ^*** indicates p<0.001, ^** indicates p<0.01, and ^* indicates p<0.05, respectively. 17 is a diagram showing that separation of lysophospholipid phosphate positional isomers is possible by column chromatography. In each graph, the horizontal axis represents retention time, and the vertical axis represents relative signal intensity when the peak top signal intensity (cps) is taken as 100%. The left column shows, from the top, the results of 17:0 lysoPI(3)P, lysoPI(4)P, and lysoPI(5)P. The right column shows, from the top, the results of 17:0 lysoPI(3,5)P2, lysoPI(3,4)P2, and lysoPI(4,5)P2.

Hereinafter, the present invention will be described with reference to the best mode. Throughout this specification, singular expressions should be understood to include the concept of the plural form, unless otherwise specified. Therefore, singular articles (e.g., in the case of English, "a", "an", "the", etc.) should be understood to include the concept of the plural form, unless otherwise specified. In addition, it should be understood that the terms used in this specification are used in the sense commonly used in the field, unless otherwise specified. Therefore, unless otherwise defined, all technical terms and scientific and technical terms used in this specification have the same meaning as commonly understood by those skilled in the art to which this invention belongs. In the event of a conflict, this specification (including definitions) will take precedence.

Below, we provide definitions of terms specifically used in this specification and/or provide an explanation of basic technical content as appropriate.

(Phospholipids)
The present invention provides novel phospholipids, as well as techniques for separating subclasses of phospholipids, phosphoinositides and lysophosphoinositides, and for measuring, detecting or identifying them.

As used herein, "phosphatidylinositol phosphate" or "phosphatidylinositol phosphate" are used interchangeably and refer to glycerin (also called propane-1,2,3-triol or glycerol) in which acyl groups are bound to the sn-1 and sn-2 positions and inositol bound to the sn-3 position is phosphorylated by X. One, two or three phosphate groups can be bound, in which case the term is referred to as "phosphatidylinositol X phosphate" (X is one, two or three). As used herein, the various phosphatidylinositol X phosphates are collectively referred to as "phosphoinositides," "phosphatidylinositol phosphates," or "PIPs." The phosphate group can be designated by its position on the inositol, e.g., phosphatidylinositol-3-monophosphate, phosphatidylinositol-4-monophosphate, phosphatidylinositol-5-monophosphate, phosphatidylinositol-3,4-diphosphate, phosphatidylinositol-3,5-diphosphate, phosphatidylinositol-4,5-diphosphate, phosphatidylinositol-3,4,5-triphosphate, etc.

In the present invention, for the first time, a method for identifying the position of the phosphate group of PIPs while retaining the acyl group is provided. In the present invention, it has been shown that detailed information on the phosphate group of PIPs in a living body can be understood by using the identification method provided by the present invention. In particular, since the information on the phosphate group of PIPs can be analyzed while retaining the acyl group, the present invention has the potential to provide detailed information that could not be analyzed by conventional techniques in the sense that information in a living body can be truly analyzed. In the present invention, unless otherwise specified, inositol is myo-inositol, and the bond type of fatty acid is an ester type. PIPs having any acyl group and phosphate group position (on inositol) can be produced, and any type of PIP can be produced by, for example, Stuart J. Conway et al., Org. Biomol. Chem., 2010, 8, 66-76. Until now, phosphoinositides have been analyzed by separating RI-labeled or non-labeled deacylated glycerophosphoinositides, or by using molecular probes or antibodies with specific binding proteins. In that sense, there is still no analytical method that can measure intact phosphoinositides. Although an analytical method using RPLC-MS/MS has been reported for PIP3, a type of phosphoinositide, this method cannot separate PIP1 and PIP2, which have three isomers, and the total of the three isomers is measured.

In this specification, the terms "lysophosphatidylinositol phosphate" or "lysophosphatidylinositol phosphate" are used interchangeably and refer to a phosphatidylinositol in which one acyl group has been removed and the inositol attached to the sn-3 position has been phosphorylated by X phosphorylations. One, two or three phosphate groups can be attached, in which case it is referred to as "lysophosphatidylinositol X phosphate" (X is one, two or three). In this specification, various lysophosphatidylinositol X phosphates are collectively referred to as "lysophosphoinositides", "lysophosphatidylinositol phosphates" or "LPIPs". The phosphate groups can be indicated by the position on the inositol, for example, lysophosphatidylinositol-3,4,5-triphosphate. In the present invention, a method for identifying this LPIPs is provided for the first time, and a method for synthesizing it is also provided. In the present invention, the use of the identification method provided by the present invention has demonstrated that LPIPs also exist in living organisms. Furthermore, the positions of phosphate groups in LPIPs could be identified in the same manner as in PIPs. In the present invention, unless otherwise specified, inositol is myo-inositol, and the bond type of fatty acid is an ester type.

In this specification, the term "sn-" is an abbreviation for Stereospecifically Numbered, and is used when the carbon atoms of a glycerin derivative are stereospecifically numbered. When the glycerin derivative is racemic, rac- is added, and when the stereochemistry is unknown, X- is added. There are two types of LPIPs, sn-1-LPIPs and sn-2-LPIPs, depending on the bonding position of the acyl group. For actual indication, the position of the acyl group is used as is, such as 1-acyl-phosphatidylinositol X phosphate and 2-acyl-phosphatidylinositol X phosphate. When there is no information about sn in this specification, it is used without any particular distinction or as a generic term (higher concept).

Once the desired fatty acid is available, it can be used to produce the corresponding PIPs and applied to the synthesis method of the present invention to produce LPIPs having the desired fatty acid group.

Specific embodiments of the present invention include the following.
monophosphate, sn-1 position 1-butanyl-lysophosphatidylinositol 4-monophosphate;
1-Hexanyl-lysophosphatidylinositol 4-monophosphate;
1-octanyl-lysophosphatidylinositol 4-monophosphate;
1-Hexadecanyl-lysophosphatidylinositol 4-monophosphate <palmityl>;
1-9Z-hexadecenyl-lysophosphatidylinositol 4-monophosphate <palmitrenyl>;
1-heptadecanyl-lysophosphatidylinositol 4-monophosphate;
1-Octadecanyl-lysophosphatidylinositol 4-monophosphate <stearyl>;
1-9Z-Octadecenyl-lysophosphatidylinositol 4-monophosphate <oleinyl>;
1-9Z,12Z-octadecanedienyl-lysophosphatidylinositol 4-monophosphate <linoleic acid>;
1-6Z,9Z,12Z-octadecanedienyl-lysophosphatidylinositol 4-monophosphate <gamma linolenic acid>;
1-8Z,11Z,14Z-icosatrienyl-lysophosphatidylinositol 4-monophosphate;
1-5Z,8Z,11Z,14Z-icosatetraenyl-lysophosphatidylinositol 4-monophosphate <arachidonic acid>;
1-7Z,10Z,13Z,16Z-docosatetraenyl-lysophosphatidylinositol 4-monophosphate;
1-7Z,10Z,13Z,16Z,19Z-docosapentaenyl-lysophosphatidylinositol 4-monophosphate;
1-4Z,7Z,10Z,13Z,16Z,19Z-docosahexaenyl-lysophosphatidylinositol 4-monophosphate;
monophosphate, sn-2 position 2-butanyl-lysophosphatidylinositol 4-monophosphate;
2-Hexanyl-lysophosphatidylinositol 4-monophosphate;
2-octanyl-lysophosphatidylinositol 4-monophosphate;
2-Hexadecanyl-lysophosphatidylinositol 4-monophosphate <palmityl>;
2-9Z-hexadecenyl-lysophosphatidylinositol 4-monophosphate <palmitrenyl>;
2-heptadecanyl-lysophosphatidylinositol 4-monophosphate;
2-Octadecanyl-lysophosphatidylinositol 4-monophosphate <stearyl>;
2-9Z-Octadecenyl-lysophosphatidylinositol 4-monophosphate <oleinyl>;
2-9Z,12Z-octadecanedienyl-lysophosphatidylinositol 4-monophosphate (linoleic acid);
2-6Z,9Z,12Z-octadecanedienyl-lysophosphatidylinositol 4-monophosphate <gamma linolenic acid>;
2-8Z,11Z,14Z-icosatrienyl-lysophosphatidylinositol 4-monophosphate;
2-5Z,8Z,11Z,14Z-icosatetraenyl-lysophosphatidylinositol 4-monophosphate <arachidonic acid>;
2-7Z,10Z,13Z,16Z-docosatetraenyl-lysophosphatidylinositol 4-monophosphate;
2-7Z,10Z,13Z,16Z,19Z-docosapentaenyl-lysophosphatidylinositol 4-monophosphate;
2-4Z,7Z,10Z,13Z,16Z,19Z-docosahexaenyl-lysophosphatidylinositol 4-monophosphate;
diphosphate, sn-1 position 1-butanyl-lysophosphatidylinositol 4,5-diphosphate;
1-Hexanyl-lysophosphatidylinositol 4,5-bisphosphate;
1-octanyl-lysophosphatidylinositol 4,5-bisphosphate;
1-Hexadecanyl-lysophosphatidylinositol 4,5-bisphosphate <palmityl>;
1-9Z-hexadecenyl-lysophosphatidylinositol 4,5-bisphosphate <palmitrenyl>;
1-heptadecanyl-lysophosphatidylinositol 4,5-bisphosphate;
1-Octadecanyl-lysophosphatidylinositol 4,5-bisphosphate <stearyl>;
1-9Z-Octadecenyl-lysophosphatidylinositol 4,5-bisphosphate <oleinyl>;
1-9Z,12Z-octadecanedienyl-lysophosphatidylinositol 4,5-bisphosphate (linoleic acid);
1-6Z,9Z,12Z-octadecanedienyl-lysophosphatidylinositol 4,5-bisphosphate <gamma linolenic acid>;
1-8Z,11Z,14Z-icosatrienyl-lysophosphatidylinositol 4,5-bisphosphate;
1-5Z,8Z,11Z,14Z-icosatetraenyl-lysophosphatidylinositol 4,5-bisphosphate <arachidonic acid>;
1-7Z,10Z,13Z,16Z-docosatetraenyl-lysophosphatidylinositol 4,5-bisphosphate;
1-7Z,10Z,13Z,16Z,19Z-docosapentaenyl-lysophosphatidylinositol 4,5-bisphosphate;
1-4Z,7Z,10Z,13Z,16Z,19Z-docosahexaenyl-lysophosphatidylinositol 4,5-bisphosphate;
diphosphate, sn-2 position 2-butanyl-lysophosphatidylinositol 4,5-diphosphate;
2-Hexanyl-lysophosphatidylinositol 4,5-bisphosphate;
2-octanyl-lysophosphatidylinositol 4,5-bisphosphate;
2-Hexadecanyl-lysophosphatidylinositol 4,5-bisphosphate <palmityl>;
2-9Z-hexadecenyl-lysophosphatidylinositol 4,5-bisphosphate <palmitrenyl>;
2-heptadecanyl-lysophosphatidylinositol 4,5-bisphosphate;
2-Octadecanyl-lysophosphatidylinositol 4,5-bisphosphate <stearyl>;
2-9Z-Octadecenyl-lysophosphatidylinositol 4,5-bisphosphate <oleinyl>;
2-9Z,12Z-octadecanedienyl-lysophosphatidylinositol 4,5-bisphosphate (linoleic acid);
2-6Z,9Z,12Z-octadecanedienyl-lysophosphatidylinositol 4,5-bisphosphate <gamma linolenic acid>;
2-8Z,11Z,14Z-icosatrienyl-lysophosphatidylinositol 4,5-bisphosphate;
2-5Z,8Z,11Z,14Z-icosatetraenyl-lysophosphatidylinositol 4,5-bisphosphate <arachidonic acid>;
2-7Z,10Z,13Z,16Z-docosatetraenyl-lysophosphatidylinositol 4,5-bisphosphate;
2-7Z,10Z,13Z,16Z,19Z-docosapentaenyl-lysophosphatidylinositol 4,5-bisphosphate;
2-4Z,7Z,10Z,13Z,16Z,19Z-docosahexaenyl-lysophosphatidylinositol 4,5-bisphosphate;
Triphosphate, sn-1 position 1-butanyl-lysophosphatidylinositol 3,4,5-triphosphate;
1-Hexanyl-lysophosphatidylinositol 3,4,5-triphosphate;
1-octanyl-lysophosphatidylinositol 3,4,5-triphosphate;
1-Hexadecanyl-lysophosphatidylinositol 3,4,5-triphosphate <palmityl>;
1-9Z-hexadecenyl-lysophosphatidylinositol 3,4,5-triphosphate <palmitrenyl>;
1-heptadecanyl-lysophosphatidylinositol 3,4,5-triphosphate;
1-Octadecanyl-lysophosphatidylinositol 3,4,5-triphosphate <stearyl>;
1-9Z-Octadecenyl-lysophosphatidylinositol 3,4,5-triphosphate <oleinyl>;
1-9Z,12Z-octadecanedienyl-lysophosphatidylinositol 3,4,5-triphosphate <linoleic acid>;
1-6Z,9Z,12Z-octadecanedienyl-lysophosphatidylinositol 3,4,5-triphosphate <gamma linolenic acid>;
1-8Z,11Z,14Z-icosatrienyl-lysophosphatidylinositol 3,4,5-triphosphate;
1-5Z,8Z,11Z,14Z-icosatetraenyl-lysophosphatidylinositol 3,4,5-triphosphate <arachidonic acid>;
1-7Z,10Z,13Z,16Z-docosatetraenyl-lysophosphatidylinositol 3,4,5-triphosphate;
1-7Z,10Z,13Z,16Z,19Z-docosapentaenyl-lysophosphatidylinositol 3,4,5-triphosphate;
1-4Z,7Z,10Z,13Z,16Z,19Z-docosahexaenyl-lysophosphatidylinositol 3,4,5-triphosphate;
Triphosphate, sn-2 position 2-butanyl-lysophosphatidylinositol 3,4,5-triphosphate;
2-Hexanyl-lysophosphatidylinositol 3,4,5-triphosphate;
2-octanyl-lysophosphatidylinositol 3,4,5-triphosphate;
2-Hexadecanyl-lysophosphatidylinositol 3,4,5-triphosphate <palmityl>;
2-9Z-hexadecenyl-lysophosphatidylinositol 3,4,5-triphosphate <palmitrenyl>;
2-heptadecanyl-lysophosphatidylinositol 3,4,5-triphosphate;
2-Octadecanyl-lysophosphatidylinositol 3,4,5-triphosphate <stearyl>;
2-9Z-Octadecenyl-lysophosphatidylinositol 3,4,5-triphosphate <oleinyl>;
2-9Z,12Z-octadecanedienyl-lysophosphatidylinositol 3,4,5-triphosphate <linoleic acid>;
2-6Z,9Z,12Z-octadecanedienyl-lysophosphatidylinositol 3,4,5-triphosphate <gamma linolenic acid>;
2-8Z,11Z,14Z-icosatrienyl-lysophosphatidylinositol 3,4,5-triphosphate;
2-5Z,8Z,11Z,14Z-icosatetraenyl-lysophosphatidylinositol 3,4,5-triphosphate <arachidonic acid>;
2-7Z,10Z,13Z,16Z-docosatetraenyl-lysophosphatidylinositol 3,4,5-triphosphate;
2-7Z,10Z,13Z,16Z,19Z-docosapentaenyl-lysophosphatidylinositol 3,4,5-triphosphate;
2-4Z,7Z,10Z,13Z,16Z,19Z-docosahexaenyl-lysophosphatidylinositol 3,4,5-trisphosphate It is understood, based on the present disclosure, that these fatty acids or fatty acid-based acyl groups are available in the lysophosphatidylinositol phosphates of the present invention in either sn-1 or sn-2 attached form.

In this specification, the term "acyl (group)" is used in the usual sense in the art and refers to a group formed by removing a hydroxyl group from an organic acid (carboxylic acid; fatty acid). In a broad sense, it includes formyl group HCO-, acetyl group CH ₃ CO-, malonyl group -COCH ₂ CO-, benzoyl group C ₆ H ₅ CO-, cinnamoyl group C ₆ H ₅ CH=CHCO-, and the like, as well as ketone derivatives. In a preferred embodiment, the acyl groups contained in phosphatidylinositol phosphate and lysophosphatidylinositol phosphates are also referred to as fatty acid groups because they form fatty acids. In this specification, fatty acids can be represented by the number of carbon atoms and the number of double bonds, and for example, arachidonic acid can be represented as (20:4). When the position of the double bond is further specified, all positions can be specified for the indication of cis or trans, or the indication of E or Z, or can be represented by a system such as the ω3 system or the ω6 system.

In the present specification, the following fatty acids and acyl groups based thereon are generally used, but it is understood that fatty acids having any chain length and any double bond can be used without being limited thereto. For example, in terms of the number of carbon atoms, those having 1 or more, typically 1 to 30, and usually 4 to 30, can be mentioned, but are not limited to, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, etc. Furthermore, the number of double bonds can be any number that is allowable depending on the number of carbon atoms, such as 0, 1, 2, 3, 4, 5, 6, 7, etc. The positions of the double bonds are typically ω-3, ω-6, ω-9, etc., and ω-5 and ω-7 have also been confirmed, and any available one of these can be used. The fatty acid may contain triple bonds, and any allowable number depending on the number of carbon atoms, such as 0, 1, 2, 3, 4, 5, 6, 7, etc., can be used.

(Separation techniques: chromatography, mass spectrometry, ion mobility separation)
"Chromatography" is used herein in its usual sense in the art to refer to the process of selective retardation of one or more components in a medium as the medium passes uniformly through a column or capillary passage of material. Retardation occurs due to the distribution of mixture components between the stationary phase(s) and the bulk medium (i.e., mobile phase) as the medium moves relative to the stationary phase(s). Examples of "chromatography" include liquid chromatography (LC) and gas chromatography (GC). In one embodiment, chromatography can be used to identify the attachment position of a phosphate group.

As used herein, "gas chromatography" or "GC" is used in its ordinary sense in the art to mean a process in which one or more components in a gas are selectively retarded as the gas passes uniformly through a column or capillary passage of material. Retardation occurs due to the distribution of mixture components between the bulk gas (i.e., mobile phase) and the stationary phase(s) as the gas moves relative to the stationary phase(s).

As used herein, "liquid chromatography" or "LC" is used in its ordinary sense in the art to refer to a process in which one or more components of a fluid solution are selectively retarded as the fluid passes uniformly through a column or capillary passage of material (permeation). Retardation occurs due to the distribution of mixture components between the bulk fluid (i.e., mobile phase) and the stationary phase(s) as the fluid moves relative to the stationary phase(s). Examples of "liquid chromatography" include reversed-phase liquid chromatography (RPLC), ion chromatography (IC), high-performance liquid chromatography (HPLC), and turbulent flow liquid chromatography (TFLC) (also referred to as highly turbulent liquid chromatography (HTLC) or high-throughput liquid chromatography). As used herein, the term "high-performance liquid chromatography" or "HPLC" (also referred to as "high-pressure liquid chromatography") refers to liquid chromatography in which separation is enhanced by forcing the mobile phase under pressure through a stationary phase, typically a densely packed column. In a preferred embodiment, HPLC is utilized. The separation mode of the packing material includes normal phase, reverse phase, partition, chiral, ion exchange, molecular sieve, affinity column, etc., and any column chromatography that separates hydrophilic groups and hydrophobic groups can be used, but among them, chiral, reverse phase, normal phase, and ion exchange columns are preferred. In one preferred embodiment, the present invention shows that by using a chiral column, all of the binding positions of phosphate groups can be identified without using other columns.

Examples of chiral columns include, but are not limited to, polysaccharide derivative chiral columns, protein-bonded chiral columns, chemically bonded optically active crown ether chiral columns, crown ether chiral columns, zwitterionic molecular columns, anion exchange chiral columns, ligand exchange chiral columns, polymethacrylate chiral columns, and polysaccharide derivative chiral columns (e.g., those sold by Daicel Corporation (Osaka, Japan)).

In liquid chromatography analysis, a sample or components of a sample may be applied to an LC column at an inlet, eluted with a solvent or solvent mixture, and discharged at an outlet. Various solvent modes may be selected to elute the analyte(s) of interest. For example, liquid chromatography may be performed using gradient, isocratic, or polymorphic (i.e., mixed) modes. During chromatography, separation of materials is influenced by variables such as the choice of eluent (also called the "mobile phase"), elution mode, gradient conditions, temperature, etc.

In this specification, "ion mobility separation (IMS)" refers to a technique for separating ionized compounds according to their bulkiness (collision cross section), and is used alone or in combination with mass spectrometry or chromatography. It is a technique for separating ions according to their mobility, developed in the 1950s, and is widely used for detecting explosives. In IMS, separation is typically performed according to the mobility determined by the collision of ions with neutral gas molecules when ions move in a gas cell at a relatively high pressure (from atmospheric pressure to several mbar) due to an electric field. As an example, DMS type (Differential ion Mobility Spectrometry) may also be used. In an exemplary embodiment, when gaseous ions pass through an IMS cell filled with _N2 gas, the bulkier the compound, the more frequently it will collide with _N2 molecules, and the mobility will decrease. Compounds can be separated based on the resulting difference in drift time. By combining with mass spectrometry (MS), it is possible to perform separation analysis of components with the same molecular weight and analysis of the three-dimensional structure of ions.

In this specification, "mass spectrometry" or "MS" is used in the usual sense in the field, and refers to an analytical method for identifying compounds by their mass. It refers to a technique in which particles such as atoms, molecules, and clusters are converted into gaseous ions (ionization) by some method, and the ions are moved in a vacuum and separated and detected according to their mass-to-charge ratio using electromagnetic forces or flight time differences. MS refers to a method of filtering, detecting, and measuring ions based on their mass-to-charge ratio, i.e., "m/z". With the recent dramatic improvement in detection sensitivity and mass resolution, the range of applications is expanding and it is being used in many fields. Representative methods are shown in Clark J et al., Nat Methods. 2011 March; 8(3): 267-272.doi:10.1038/nmeth.1564. MS techniques generally include the steps of (1) ionizing compounds to form charged compounds; and (2) detecting the molecular weight of the charged compounds and calculating the mass-to-charge ratio. Compounds may be ionized and detected by suitable means. A "mass spectrometer" generally includes an ionizer, a mass analyzer, and an ion detector. In general, one or more molecules of interest are ionized, and the ions are then introduced into a mass spectrometry instrument, where the ions follow a path in space that depends on their mass ("m") and charge ("z") due to a combination of magnetic and electric fields. For a review of mass spectrometers, see, for example, Jurgen H, "Mass Spectrometry", Maruzen Publishing (2014). Mass spectrometers include, for example, magnetic, quadrupole, and time-of-flight types, but it is preferable to use a quadrupole type that has good quantitative performance, a wide dynamic range, and good linearity. Detection of ions in quantification includes selected ion monitoring, which selectively detects only the ions of interest, and selected reaction monitoring (SRM), which selects one of the ion species purified in the first mass analyzer as a precursor ion and detects product ions generated by the fragmentation of the precursor ion in the second mass analyzer. SRM improves the signal/noise ratio by increasing selectivity and reducing noise.

As used herein, the term "resolution" or "resolution (FWHM)" (also known in the art as "m/Δm50%") refers to the observed mass-to-charge ratio divided by the width of the mass peak at 50% of its maximum height (full width half maximum, "FWHM"). In the present invention, it is preferable to use an analyzer with high resolution. Higher resolution can result in better qualitative and quantitative results.

In a practical arrangement, by careful selection of valves and connector tubing, two or more chromatography columns may be connected as needed so that material passes from one to the other without the need for any manual steps. In a preferred embodiment, the selection of valves and tubing is controlled by a computer preprogrammed to perform the necessary steps. Most preferably, the chromatography system is also connected in such an on-line manner to a detection system, e.g., an MS system. Thus, an operator may place a tray of samples into the autosampler and the remaining operations are performed under computer control, resulting in purification and analysis of all selected samples.

In this specification, "chromatography-mass spectrometry (C-MS)" refers to an analytical method performed using an apparatus that combines a chromatograph (e.g., a gas chromatograph, liquid chromatograph, etc.) with a mass spectrometer. For example, tandem mass spectrometry (C-MS/MS) in which multiple mass spectrometry units are combined can also be used. When ionization is performed in C-MS, for example, atmospheric pressure chemical ionization, ESI, atmospheric pressure photoionization, etc. can be used.

In this specification, "liquid column chromatography-mass spectrometry (LC-MS)" refers to an analytical method performed using an apparatus that combines a liquid chromatograph and a mass spectrometer. For example, tandem mass spectrometry (LC-MS/MS), in which multiple mass spectrometry units are combined, can also be used. When performing ionization in LC-MS, for example, atmospheric pressure chemical ionization, ESI, atmospheric pressure photoionization, etc. can be used.

In this specification, "ion mobility separation-mass spectrometry (IMS-MS)" refers to an analytical method performed using an apparatus that combines ion mobility separation (IMS) and a mass spectrometer. For example, tandem mass spectrometry (IMS-MS/MS) in which multiple mass spectrometry units are combined can also be used. When performing ionization in IMS-MS, for example, atmospheric pressure chemical ionization, ESI, atmospheric pressure photoionization, etc. can be used. IMS-MS enables the separation of interfering components with the same m/z, which is not possible with a mass spectrometer alone, and the analysis of the three-dimensional structure of ions, providing scientists with more specific and detailed information.

As used herein, "measurement" is used in its usual sense in the field, and refers to measuring and determining the amount of a certain object. As used herein, "detection" is used in its usual sense in the field, and refers to testing and finding a substance or component, etc., "identification" refers to the act of searching for an object's classification within existing categories related to that object, and when used in the field of chemistry, refers to determining the identity of the target substance as a chemical substance (e.g., determining its chemical structure), and "quantitation" refers to determining the amount of the target substance present.

In this specification, the term "run" is used in the usual sense as used in the art, and refers to a series of steps from loading a sample into a separation means such as mass spectrometry, chromatography, or ion mobility separation, separating the components in the sample, and washing as necessary. Usually, different samples are measured in different runs to avoid confusion. When there are multiple objects to be measured, it is advantageous to be able to measure all of the objects to be measured in the same run, but the run may be divided into multiple runs taking into account the adaptability of each object to the measurement system, dynamic range, and resolution of each object to be measured in the sample.

As used herein, the "amount" of an analyte in a bodily fluid sample generally refers to an absolute value reflecting the mass of analyte that can be detected in a volume of the sample. However, amount also contemplates a relative amount compared to the amount of another analyte. For example, the amount of an analyte in a sample may be an amount that is greater than a control or normal level of the analyte that is normally present in the sample.

The term "about," when used herein in connection with quantitative measurements that do not include measurement of the mass of an ion, refers to the indicated value plus or minus 10%. Mass spectrometry instruments may vary slightly in determining the mass of a given analyte. The term "about," in connection with the mass of an ion or the mass/charge ratio of an ion, refers to +/- 0.5 atomic mass units.

As used herein, the term "alkylamine" refers to an alkyl group having an amine (NH ₂ --) group bonded thereto. Representative examples include, but are not limited to, methylamine, ethylamine, propaneamine, and the like. They are also called aliphatic amines or aminoalkanes.

In this specification, the term "anion exchange resin" is used in the usual sense in the relevant field, and typically refers to a resin that has basic groups bonded to its surface and has the property of binding with anions, allowing the concentration of anions, etc.

In this specification, "acidic phospholipid" refers to a phospholipid in which the negative charge of the phosphate group is not neutralized by a base, such as phosphatidic acid (PA), phosphatidylserine (PS), and phosphatidylinositol (PI).

In this specification, the "protecting group" of the phosphate group may be any group used in the art. Examples of the protecting group are given in Clark J et al., Nat Methods. 2011 March;8(3):267-272.doi:10.1038/nmeth.1564. Examples of the protecting group include benzyl, p-methoxybenzyl, and tert-butyl.

As used herein, "conditions under which phosphatidylinositol phosphate and/or lysophosphatidylinositol phosphate are not decomposed" refers to conditions under which components such as acyl groups are not decomposed and released from phosphatidylinositol phosphate and/or lysophosphatidylinositol phosphate. "Conditions under which phosphatidylinositol phosphate is not decomposed" refers to conditions under which components such as acyl groups are decomposed and not released from phosphatidylinositol phosphate, "conditions under which lysophosphatidylinositol phosphate is not decomposed" refers to conditions under which components such as acyl groups are decomposed and not released from lysophosphatidylinositol phosphate, and "conditions under which phosphatidylinositol phosphate and lysophosphatidylinositol phosphate are not decomposed" refers to conditions under which components such as acyl groups are decomposed and not released from phosphatidylinositol phosphate and lysophosphatidylinositol phosphate. Such conditions include applying the sample directly to chromatography, ion mobility separation, or MS, as well as performing other treatments under conditions that do not include deacylation treatment to prepare a sample that can be applied to chromatography, ion mobility separation, or MS, and also include, for example, not performing a treatment that denatures the sample and performing a treatment that introduces a protecting group into the phosphate group.

As used herein, "without deacylation treatment" refers to not performing deacylation, for example, treatment with phospholipase A (phospholipase _A1 , _A2 , etc.) or treatment in the presence of an alkylamine such as methylamine (see Serunian, et al., Methods in Enzymol. Vol., 198, 1991, pp. 78-87, especially pp. 82-83, and Fujii et al. Folia Pharmacol Jpn. Vol. 142, 2013, pp. 236-240, especially pp. 238-239, which disclose deacylation conditions).

As used herein, "intact (sample)" refers to a sample that is not denatured when considering a sample to be analyzed. Denaturation of a sample can be exemplified by denaturation caused by heating, oxidation caused by the action of oxygen in the air, moisture, heat, light, metal ions, microorganisms or enzymes, and hydrolysis in an aqueous solvent. In order to keep a sample intact, for example, the sample can be promptly frozen after preparation and stored at -80°C until further manipulation is performed.

In this specification, "unlabeled" and "without labeling" refer to the absence of a label on the sample to be analyzed. In this specification, "label" refers to an entity (e.g., a substance, energy, electromagnetic waves, etc.) that distinguishes the target molecule or substance from others. Examples of such labeling methods include RI (radioisotope) method, fluorescence method, biotin method, chemiluminescence method, etc. When multiple markers of the present invention or factors or means for capturing them are labeled by a fluorescence method, the labeling is performed with fluorescent substances that have different maximum fluorescence emission wavelengths. In the present invention, such labels can be used to modify the target object so that it can be detected by the detection means used. Such modifications are known in the art, and those skilled in the art can carry out such methods appropriately depending on the label and the target object.

In this specification, "diagnosis" refers to identifying various parameters related to a disease, disorder, condition (e.g., a disease, disorder, condition, etc. caused by lipid mediators) in a subject, and judging the current or future state of such a disease, disorder, or condition. By using the method, device, and system of the present invention, the internal state can be examined, and such information can be used to select various parameters such as a disease, disorder, condition, and a treatment or prevention formulation or method to be administered in the subject. In the narrow sense of this specification, "diagnosis" refers to diagnosing the current state, but in the broad sense, it includes "early diagnosis," "predictive diagnosis," "pre-diagnosis," etc. The diagnostic method of the present invention is industrially useful because, in principle, it can utilize something that comes out of the body and can be performed without the hands of a medical professional such as a doctor. In this specification, in order to clarify that it can be performed without the hands of a medical professional such as a doctor, it is sometimes referred to as "supporting" "predictive diagnosis, pre-diagnosis, or diagnosis". The technology of the present invention is applicable to such diagnostic technology. The techniques of the present invention can be used to identify the presence of phosphoinositides and/or lysophosphoinositides or the location of their phosphate groups for a variety of such diagnostic applications.

In this specification, "treatment" refers to preventing, preferably maintaining, more preferably alleviating, and even more preferably eradicating a disease or disorder (e.g., a disorder such as cancer, a disease or disorder caused by a lipid mediator, etc.) from worsening when the disease or disorder has progressed, and includes exerting a symptom-improving or preventive effect on the patient's disease or one or more symptoms associated with the disease. Diagnosis in advance and appropriate treatment are called "companion treatment," and diagnostic agents for this purpose are sometimes called "companion diagnostic agents." Identifying the presence of phosphoinositides and/or lysophosphoinositides or the binding position of their phosphate groups using the technology of the present invention can be useful in such companion treatment or companion diagnosis, since it can be associated with a specific disease state.

As used herein, "prevention" refers to preventing a certain disease or disorder (e.g., cancer, diseases or disorders associated with lipid mediators, etc.) from occurring before such a state occurs. Diagnosis can be performed using the PIPs and/or LPIPs of the present invention, and, if necessary, it may be possible to prevent, for example, diseases using the agents of the present invention, or to take preventive measures. In the present specification, the term "prophylactic drug (agent)" refers, in a broad sense, to any agent that can prevent a target state (e.g., cancer, diseases or disorders associated with lipid mediators, etc.).

As used herein, the term "prognosis" refers to predicting the likelihood of death or progression due to a disorder such as cancer, a disease caused by lipid mediators, or a disorder. Prognostic factors are variables related to the natural course of a disease, and they affect the recurrence rate, etc., of a patient once the disease has developed. Clinical indicators associated with poor prognosis include, for example, any of the cellular indicators used in the present invention. Prognostic factors are often used to classify patients into subgroups with different pathologies. Identifying the presence of phosphatidylinositol and/or lysophosphatidylinositol or the binding site of the phosphate group using the technology of the present invention can be useful as a technology to provide prognostic factors, since they can be associated with specific disease states.

In this specification, the term "detection device" refers broadly to any device capable of detecting or testing a target object. In the present invention, chromatography, ion mobility separation devices, mass analyzers, etc. are also included in the detection device.

As used herein, the term "diagnostic device" refers broadly to any agent capable of diagnosing a condition of interest (e.g., cancer, diseases or disorders related to lipid mediators, etc.).

In this specification, the terms "drug," "agent," or "factor" (all of which correspond to the English term agent) are used interchangeably in a broad sense and may refer to any substance or other element (e.g., energy such as light, radioactivity, heat, electricity, etc.) as long as it can achieve the intended purpose. Examples of such substances include, but are not limited to, proteins (including antibodies, etc.), polypeptides, oligopeptides, peptides, polynucleotides, oligonucleotides, nucleotides, nucleic acids (including, for example, DNA such as cDNA and genomic DNA, and RNA such as mRNA), polysaccharides, oligosaccharides, lipids, organic small molecules (e.g., hormones, ligands, signaling substances, organic small molecules, molecules synthesized by combinatorial chemistry, small molecules that can be used as pharmaceuticals, etc.), and composite molecules thereof.

As used herein, the term "detection agent" or "test agent" refers broadly to any agent capable of detecting or testing a target.

As used herein, the term "diagnostic agent" refers broadly to any agent capable of diagnosing a target condition (e.g., cancer, a disease or disorder associated with lipid mediators, etc.).

As used herein, the term "therapeutic agent" refers broadly to any drug capable of treating a target condition (e.g., cancer, a disease or disorder associated with lipid mediators, etc.).

As used herein, "cancer" refers to any cancer that can be detected by the markers of the present invention, including, but not limited to, hepatocellular carcinoma, esophageal squamous cell carcinoma, breast cancer, pancreatic cancer, squamous cell carcinoma or adenocarcinoma of the head and neck, colorectal cancer, renal cancer, brain cancer (tumor), prostate cancer, small cell and non-small cell lung cancer, bladder cancer, bone or joint cancer, uterine cancer, cervical cancer, multiple myeloma, hematopoietic malignancies, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma, skin cancer, melanoma, squamous cell carcinoma, leukemia, lung cancer, ovarian cancer, gastric cancer, Kaposi's sarcoma, laryngeal cancer, endocrine cancer, thyroid cancer, parathyroid cancer, pituitary cancer, adrenal cancer, cholangiocarcinoma, endometriosis, esophageal cancer, liver cancer, NSCLC, osteosarcoma, pancreatic cancer, SCLC, soft tissue tumors, AML, and CML.

As used herein, "inflammation" refers to any inflammation that can be detected by the marker of the present invention, and may involve activation of blood cells associated with inflammation, such as macrophages (chemotaxis of inflammatory cells, production of reactive oxygen species, phagocytosis, enzyme secretion reactions, etc.). Exemplary inflammation includes, for example, arthritis, tendonitis, bursitis, psoriasis, cystic fibrosis, Sjogren's syndrome, giant cell arteritis, progressive systemic sclerosis (scleroderma), spondylitis, polymyositis, dermatomyositis, pemphigus, pemphigoid, Hashimoto's thyroiditis, cholangitis, inflammatory bowel disease (IBD, e.g., Crohn's disease, ulcerative colitis), colitis, inflammatory skin disease, pneumonia, asbestosis, silicosis, bronchiectasis, talc, pneumoconiosis, sarcoidosis, delayed hypersensitivity reactions (e.g., poison ivy dermatitis), inflammation of the airways, adult respiratory distress syndrome (ARDS), encephalitis, immediate hypersensitivity reactions, asthma, hay fever, allergies, acute anaphylaxis, reperfusion injury, rheumatoid arthritis, glomerulonephritis, pyelonephritis, cellulitis, cystitis, cholecystitis, allograft rejection, host versus lymphoma. Graft rejection, appendicitis, arteritis, blepharitis, bronchiolitis, bronchitis, cervicitis, cholangitis, chorioamnionitis, conjunctivitis, dacryoadenitis, dermatomyositis, endocarditis, endometritis, enteritis, enterocolitis, epicondylitis, epididymitis, fasciitis, fibrositis, gastritis, gastroenteritis, gingivitis, ileitis, iritis, laryngitis, myelitis, myocarditis, nephritis, omphalitis, oophoritis, orchitis, osteitis, otitis media, pancreatitis, parotid gland In some embodiments, the inflammation may be caused by inflammation of the lining of the rectum, such as inflammatory bowel disease, pericarditis, pharyngitis, pleuritis, phlebitis, pneumonia, proctitis, prostatitis, rhinitis, salpingitis, sinusitis, stomatitis, synovitis, orchitis, tonsillitis, urethritis, cystitis, uveitis, vaginitis, vasculitis, vulvitis, vulvovaginitis, vasculitis, osteomyelitis, optic neuritis, myelitis, and fasciitis.

In this specification, the term "cancer marker", also referred to as a tumor marker, refers to a biological substance or a substance found in the living body that is effective in diagnosing cancer, monitoring the progress after treatment, and detecting recurrence or metastasis. Typically, cancer can be diagnosed by measuring a substance in the blood. In this case, the substance in the blood corresponds to a cancer marker.

As used herein, the term "marker" refers to a substance that serves as an indicator for tracking whether a certain state (e.g., functionality, transformed state, disease state, disorder state, or the level, presence or absence of proliferation ability, differentiation state, etc.) is in a state or there is a risk of it being in a state. As used herein, the term "disease marker" refers to a substance that serves as an indicator for tracking whether a certain state is in a state or there is a risk of it being in a state. In the present invention, detection, diagnosis, preliminary detection, prediction, or advance diagnosis of a certain state (e.g., disease state, health state, cell or tissue differentiation disorder, etc.) can be realized using a detection method provided by the present invention, as well as a drug, agent, factor, or means specific to the marker, or a composition, kit, system, etc. containing them.

As used herein, a "purified" substance or biological factor (e.g., a DNA vaccine, a nucleic acid construct, a nucleic acid such as a plasmid DNA, or a protein) refers to a substance or biological factor from which at least a portion of the factors naturally associated with the substance or biological factor have been removed. Thus, the purity of the biological factor in a purified biological factor is usually higher (i.e., concentrated) than the state in which the biological factor is normally present. As used herein, the term "purified" means that the same type of biological factor is present in an amount of preferably at least 75% by weight, more preferably at least 85% by weight, even more preferably at least 95% by weight, and most preferably at least 98% by weight. The substance or biological factor used in the present invention is preferably a "purified" substance. As used herein, an "isolated" substance or biological factor (e.g., a nucleic acid or a protein) refers to a substance or biological factor from which factors naturally associated with the substance or biological factor have been substantially removed. The term "isolated" as used herein does not necessarily denote purity, as this can vary depending on the purpose, but, where necessary, preferably means that at least 75%, more preferably at least 85%, even more preferably at least 95%, and most preferably at least 98% by weight of the same type of biological factor is present. The materials used in the present invention are preferably "isolated" materials or biological factors.

Generally, the compositions, medicaments, agents (therapeutic agents, prophylactic agents, etc.) of the present invention comprise a therapeutically effective amount of a medicament or active ingredient, and a pharma- ceutically acceptable carrier or excipient. As used herein, "pharmaceutically acceptable" means approved by a government regulatory agency or listed in the Pharmacopoeia or other generally recognized pharmacopoeias for use in animals, and more particularly in humans.

In one embodiment of the present invention, the "patient" is primarily intended to be a human, but may also be a mammal other than a human, where applicable.

As used herein, the term "subject" refers to the subject of the prevention, treatment, etc. of the present invention, and includes humans and non-human mammals (e.g., one or more of mice, guinea pigs, hamsters, rats, mice, rabbits, pigs, sheep, goats, cows, horses, cats, dogs, marmosets, monkeys, or chimpanzees, etc.).

As used herein, the term "kit" refers to a unit in which the parts to be provided (e.g., test agents, diagnostic agents, therapeutic agents, reagents, labels, instructions, etc.) are provided, usually in two or more compartments. This kit form is preferred when the purpose is to provide a composition that should not be provided in a mixed state for reasons of stability, etc., but is preferably mixed immediately before use. Such a kit is preferably provided with instructions or instructions that describe how to use or process the parts to be provided (e.g., test agents, diagnostic agents, therapeutic agents, reagents, labels, etc.). When the kit is used as a reagent kit in this specification, the kit includes instructions, etc. that describe how to use the test agents, diagnostic agents, therapeutic agents, reagents, labels, etc. When a test device, a diagnostic device, etc. are combined, the "kit" may be provided as a "system".

In this specification, the term "instructions" refers to instructions for a physician or other user on how to use the present invention. The instructions include instructions on how to use the detection method of the present invention, how to use a diagnostic agent, or how to administer a medicine. The instructions may also include instructions on how to detect and evaluate a marker. The instructions are prepared in accordance with a format prescribed by the regulatory agency of the country in which the present invention is implemented (e.g., the Ministry of Health, Labor and Welfare in Japan, the Food and Drug Administration (FDA) in the United States, etc.), and clearly state that the instructions have been approved by the regulatory agency. The instructions are so-called package inserts, and are usually provided in paper form, but are not limited thereto, and may also be provided in the form of electronic media (e.g., a homepage provided on the Internet, e-mail, etc.).

Preferred Embodiments
The preferred embodiments of the present invention are described below. The embodiments provided below are provided for a better understanding of the present invention, and it is understood that the scope of the present invention should not be limited to the following description. Therefore, it is clear that a person skilled in the art can make appropriate modifications within the scope of the present invention in consideration of the description in this specification. It is also understood that the following embodiments of the present invention can be used alone or in combination.

<Lysophosphatidylinositol phosphates (LPIPs)>
In one aspect, the present invention provides a compound that is lyso-phosphatidylinositol monophosphate (LPIP1), lyso-phosphatidylinositol bisphosphate (LPIP2), or lyso-phosphatidylinositol trisphosphate (LPIP3). Research on phosphatidylinositol phosphates has been conducted from a relatively early stage, and a considerable amount of information has been elucidated in recent years (Sasaki et al., Medical Progress Vol. 248, No. 13, (2014) pp.1039-1049). Phosphoinositides/inositol phospholipids (PIPs) are said to constitute plasma membranes and organelle membranes, and their head groups are significantly larger than other glycerophospholipids due to their six-membered carbon ring and polyvalent phosphate group, and the highly charged PIPs metabolic system is said to be involved in a wide range of life phenomena such as proliferation, differentiation, movement, aging, and death. Therefore, the lipids of the present invention (LPIPs) themselves, which were identified for the first time in the present invention, as well as their metabolic enzymes and receptors, can be useful as targets for therapeutic drugs for various diseases and as disease markers. In addition, since lipid metabolism is one of the important research subjects, these lipids are used as reagents for experiments.

There have been no reports of the identification of lyso-forms of PIPs (LPIPs) because they could not be measured in the past. In the present invention, the inventors developed an analytical method that combines the concentration of acidic phospholipids using an anion exchange resin such as DEAE cellulose, the protection of phosphate groups with TMS diazomethane, and the SRM method using a triple quadrupole mass spectrometer. By using this method, they succeeded in finding LPIP1, LPIP2, and LPIP3 of the present invention, which have structures different from known inositol phospholipids. In addition, since there was no known method for synthesizing LPIPs, they could not be manufactured. Therefore, although they can be described according to the naming rules of general chemical substances, the existence of LPIPs as a substance has not been confirmed, and it can be said that they were unknown. In the present invention, by developing technology capable of detecting and identifying LPIPs, it has been discovered that LPIPs exist in vivo (here, those that exist in vivo are also referred to as "natural LPIPs"), and a synthesis method has also been developed. In addition to providing LPIPs that exist in vivo, it is now possible to provide LPIPs including "non-natural" LPIPs by synthesis.

The present invention has also demonstrated that LPIPs exist in vivo. For example, as shown in the Examples, they have been found in various mammalian cultured cell lines and mouse prostate cancer tissues, and it has also been revealed that certain molecular species such as LPIP3 appear to be preferentially expressed in cancer cells. In addition, some of the LPIPs found have been found to be specifically or preferentially expressed in cancer. Analysis of PIPs with a structure similar to LPIPs has been progressing, and for example, it is known that mutations and reduced expression of the tumor suppressor gene PTEN are frequent abnormalities observed in approximately half of human cancers. At present, it is believed that the change in PIP3 (a substrate of PTEN) decomposed by PTEN is related to carcinogenesis and progression, but there are few reports showing the change in PIP3 (Thanh-Trang T. Vo and David A. Fruman, Cancer Discov; 5(7); 697-700, 2015; Kofuji et al., Cancer Discov; 5(7); 730-9. 2015), and it is possible that the LPIPs of the present invention act as lipid mediators of cancer. In addition, highly sensitive measurements have revealed that LPIPs exist not only inside cells but also outside cells. Measuring these lipids in samples that are relatively easy to collect, such as blood and urine, is also useful in laboratory medicine.

Many of the changes in PIPs (e.g., metabolism, decomposition, etc.) remain unclear due to the immaturity of measurement techniques. In the present invention, LPIPs have been found as novel substances, and it has been found that some LPIPs are preferentially expressed in cancer, so that the LPIPs can be applied to medical tests and diagnoses using blood, urine, and other samples in cancer or other diseases associated with lipid mediators. It is generally known that phospholipase A (phospholipase A ₁ , A ₂ , etc.) is involved in the metabolism of phospholipids to the corresponding lyso form, but phospholipase A ₁ and A ₂ have extremely high substrate specificity and do not react with different types of phospholipids. For example, it is said that subtypes that use phosphatidylcholine as a substrate do not react with phosphatidylserine. In addition, those that use PIPs as a substrate are not known. In the present invention, lysophosphatidylinositol monophosphate, lysophosphatidylinositol diphosphate, and lysophosphatidylinositol triphosphate have been found. The "lysophosphatidylinositol monophosphate" found in vivo has a phosphate group at position 4, and the following species have been found to have fatty acids bound to the sn-1 or sn-2 positions: 16:0, 16:1 (9Z), 18:0, 18:1 (9Z), 18:2 (9Z, 12Z), 20:3 (8Z, 11Z, 14Z), 20:4 (5Z, 8Z, 11Z, 14Z), 22:4 (7Z, 10Z, 13Z, 16Z), 22:5 (7Z, 10Z, 13Z, 16Z, 19Z), and 22:6 (4Z, 7Z, 10Z, 13Z, 16Z, 19Z). The "lysophosphatidylinositol diphosphate" found in vivo has phosphate groups at the 4 and 5 positions, and the following fatty acids are found bound to the sn-1 or sn-2 positions: 16:0, 16:1 (9Z), 18:0, 18:1 (9Z), 18:2 (9Z, 12Z), 18:3 (6Z, 9Z, 12Z), 20:3 (8Z, 11Z, 14Z), 20:4 (5Z, 8Z, 11Z, 14Z). The "lysophosphatidylinositol triphosphate" found in vivo has the following fatty acids bound to the sn-1 or sn-2 positions: 16:0, 16:1 (9Z), 18:0, 18:1 (9Z), 20:4 (5Z, 8Z, 11Z, 14Z). These molecular species are similar to the fatty acid species found in PIPs, suggesting the existence of a previously unknown phospholipase _A1 or phospholipase _A2 that has specificity for phosphatidylinositol phosphates. The above-mentioned fatty acid molecular species appear to be found in both sn-1 and sn-2 in vivo, although the abundance varies.

In one embodiment, the present invention provides a compound that is 1-acyl-lysophosphatidylinositol monophosphate, 2-acyl-lysophosphatidylinositol monophosphate, 1-acyl-lysophosphatidylinositol diphosphate, 2-acyl-lysophosphatidylinositol diphosphate, 1-acyl-lysophosphatidylinositol triphosphate, or 2-acyl-lysophosphatidylinositol triphosphate. The acyl group may be any type of acyl group as long as the corresponding fatty acid is available. It has been found in the present invention that the lysophosphatidylinositol phosphates of the present invention can be produced using phosphatidylinositol phosphates as a starting material. Therefore, based on the method of Conway cited in this specification, the fatty acid obtained can be introduced into the phosphatidylinositol phosphates, and used as a starting material for the production method of the lysophosphatidylinositol phosphates of the present invention.

In one preferred embodiment, the present invention provides a compound which is 2-acyl-lysophosphatidylinositol monophosphate, 2-acyl-lysophosphatidylinositol diphosphate, 1-acyl-lysophosphatidylinositol triphosphate or 2-acyl-lysophosphatidylinositol triphosphate.

The compounds of the present invention can use lysophosphatidylinositol phosphates in which the phosphate groups are bound to any position on the inositol, and preferably those bound to one, two or three positions among positions 3, 4 and 5. Thus, preferred examples include 3-monophosphate type, 4-monophosphate type, 5-monophosphate type, 3,4-diphosphate type, 3,5-diphosphate type, 4,5-diphosphate type and 3,4,5-triphosphate type. In one embodiment, the present invention provides 1-acyl-lysophosphatidylinositol-3-monophosphate, 1-acyl-lysophosphatidylinositol-4-monophosphate, 1-acyl-lysophosphatidylinositol-5-monophosphate, 2-acyl-lysophosphatidylinositol-3-monophosphate, 2-acyl-lysophosphatidylinositol-4-monophosphate, 2-acyl-lysophosphatidylinositol-5-monophosphate, 1-acyl-lysophosphatidylinositol-3.4-di ... In accordance with the present invention, there is provided a compound which is lysophosphatidylinositol-3.5-diphosphate, 1-acyl-lysophosphatidylinositol-4.5-diphosphate, 2-acyl-lysophosphatidylinositol-3,4-diphosphate, 2-acyl-lysophosphatidylinositol-3,5-diphosphate, 2-acyl-lysophosphatidylinositol-4,5-diphosphate, 1-acyl-lysophosphatidylinositol-3,4,5-triphosphate, or 2-acyl-lysophosphatidylinositol-3,4,5-triphosphate.

In a specific embodiment, the present invention provides, by way of example, the following compounds, although the specific compounds of the present invention are not limited to the following:

(Chemical formula 1)

<Applications of LPIPs>
In another aspect, the present invention provides a composition for use as a disease marker, comprising the compound of the present invention (such as lysophosphatidylinositol phosphates). It is understood that such a compound can be adopted by combining one or more of any of the embodiments described in <Lysophosphatidylinositol phosphates (LPIPs)> in this specification. Examples of such a compound include, for example, lysophosphatidylinositol monophosphate (LPIP1), lysophosphatidylinositol diphosphate (LPIP2), or lysophosphatidylinositol triphosphate (LPIP3), and combinations thereof. In particular, it has been shown that the expression level of LPIP3 is significantly increased in cancer tissues, and the compound of the present invention can be used as an inflammation marker or cancer marker, particularly when it is a substance present in the living body, and it is understood that the synthetic product is also useful as a control or standard product for detecting an inflammation marker or cancer marker.

When the compound of the present invention is used as a cancer marker, the target cancer is not limited, but includes, for example, hepatocellular carcinoma, esophageal squamous cell carcinoma, breast cancer, pancreatic cancer, squamous cell carcinoma or adenocarcinoma of the head and neck, colorectal cancer, renal cancer, brain cancer (tumor), prostate cancer, small cell and non-small cell lung cancer, bladder cancer, bone or joint cancer, uterine cancer, cervical cancer, multiple myeloma, hematopoietic malignant tumor, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma, skin cancer, melanoma, squamous cell carcinoma, leukemia, lung cancer, ovarian cancer, gastric cancer, Kaposi's sarcoma, laryngeal cancer, endocrine cancer, thyroid cancer, parathyroid cancer, pituitary cancer, adrenal cancer, cholangiocarcinoma, endometriosis, esophageal cancer, liver cancer, NSCLC, osteosarcoma, pancreatic cancer, SCLC, soft tissue tumor, AML, and CML, but is not limited thereto, and is preferably understood to include prostate cancer, etc.

When the compound of the present invention is used as an inflammation marker, the target inflammation is not limited, but may be, for example, arthritis, tendonitis, bursitis, psoriasis, cystic fibrosis, Sjogren's syndrome, giant cell arteritis, progressive systemic sclerosis (scleroderma), spondylitis, polymyositis, dermatomyositis, pemphigus, pemphigoid, Hashimoto's thyroiditis, cholangitis, inflammatory bowel disease (IBD, e.g., Crohn's disease, ulcerative colitis), colitis, inflammatory skin disease, pneumonia, asbestosis, silicosis, bronchiectasis, talc, pneumoconiosis, sarcoidosis, delayed hypersensitivity reaction (e.g., poison ivy dermatitis), inflammation of the airway, adult respiratory distress syndrome (ARDS), encephalitis, immediate hypersensitivity reaction, asthma, hay fever, allergy, acute anaphylaxis, reperfusion injury, rheumatoid arthritis, glomerulonephritis, pyelonephritis, cellulitis, cystitis, cholecystitis. , allograft rejection, host-versus-graft rejection, appendicitis, arteritis, blepharitis, bronchiolitis, bronchitis, cervicitis, cholangitis, chorioamnionitis, conjunctivitis, dacryoadenitis, dermatomyositis, endocarditis, endometritis, enteritis, enterocolitis, epicondylitis, epididymitis, fasciitis, fibrositis, gastritis, gastroenteritis, gingivitis, ileitis, iritis, laryngitis, myelitis, myocarditis, nephritis, omphalitis, oophoritis, orchitis, osteitis, middle ear In some embodiments, the inflammation may be caused by inflammation of the lining of the rectum, such as, but not limited to, inflammation of the lining of the rectum, inflammation of the pancreas, inflammation of the parotitis gland, inflammation of the pericarditis, inflammation of the pharyngitis, inflammation of the pleuropneumonia, inflammation of the phlebitis, inflammation of the phlebitis, inflammation of the pneumonia, inflammation of the proctitis, inflammation of the prostate, inflammation of the rectum, inflammation of the salpingitis, inflammation of the sinusitis, inflammation of the stomatitis, inflammation of the synovitis, inflammation of the orchitis, inflammation of the tonsillitis, inflammation of the urethritis, inflammation of the cystitis, inflammation of the uveitis, inflammation of the vaginitis, inflammation of the vasculitis, inflammation of the vulvulvovaginitis, inflammation of the vasculitis, inflammation of the osteomyelitis, inflammation of the optic neuritis, inflammation of the myelitis, and inflammation of the fasciitis.

When used as a disease marker such as an inflammation marker or a cancer marker, the LPIPs of the present invention can be detected by the detection method disclosed herein or other methods (e.g., immunological methods, cytological methods using radioactive substances, etc.). When using a radioactive substance, it is assumed that intracellular LPIPs labeled with a radioisotope (RI) are separated by high performance liquid chromatography (HPLC) to obtain radioactivity, following the method for PIPs (see Sasaki et al., Medical Progress Vol. 248, No. 13, (2014) pp.1039-1049).

Detection, identification, quality control, etc. of the LPIPs of the present invention can be achieved by using the detection method described in the present invention, as well as by using a substance or an interacting molecule that binds to the marker LPIPs. In the context of the present invention, a "substance that binds" or an "interacting molecule" to a marker substance is a molecule or substance that at least temporarily binds to a molecule such as a marker substance (e.g., LPIPs) and preferably indicates that it has been bound (e.g., is labeled or can be labeled). Substances that bind to molecules such as LPIPs can be receptors or transferases (if known) for molecules such as LPIPs, and other examples include antibodies, antisense oligonucleotides, siRNA, low molecular weight molecules (LMW), binding peptides, aptamers, ribozymes, and peptidomimetics, including binding proteins specific for molecules such as LPIPs. As used herein, the term "binding protein" in reference to molecules such as LPIPs refers to a type of protein that binds (preferably specifically) to molecules such as LPIPs, and includes, but is not limited to, antibodies such as polyclonal or monoclonal antibodies, antibody fragments, and protein scaffolds raised against molecules such as LPIPs. Representative examples of such proteins include antibodies, which can be produced using techniques known in the art.

<Method for producing LPIPs>
In another aspect, the present invention relates to a method for producing the compounds of the present invention (lysophosphatidylinositol phosphates), comprising the step of deacylating phosphatidylinositol monophosphate, phosphatidylinositol diphosphate, or phosphatidylinositol triphosphate by contacting them with an alkylamine.

In a preferred embodiment, the alkylamine is methylamine.

In a further preferred embodiment, the deacylation is carried out by incubating for an appropriate period of time in the presence of an alkylamine such as methylamine at an appropriate concentration (e.g., 10.7% or less) at a temperature that promotes deacylation (e.g., room temperature to about 80°C, typically about 53°C). (See Serunian, et al., Methods in Enzymol. Vol., 198, 1991, pp. 78-87, especially pp. 82-83, and Fujii et al. Folia Pharmacol Jpn. Vol. 142, 2013, pp. 236-240, especially pp. 238-239, which disclose dediacylation conditions. These documents disclose only conditions under which both sn-1 and sn-2 acyl groups are cleaved, and may be referred to as "dediacylation" in this specification.). For example, under these conditions, the reaction is carried out under conditions of about 10.7% methylamine concentration and 50 to 120 minutes. In the present invention, by changing the conditions of Serunian and Fujii (dediacylation conditions) in a more lenient direction, unexpectedly, conditions were found under which only one of the sn-1 or sn-2 acyl groups was cleaved (referred to as "monoacyl removal" in this specification). In the present invention, it was found that, if the methylamine concentration is 10.7%, which is the same as the conditions of Serunian and Fujii, by subjecting the reaction to a short period of time, such as about 30 minutes or less, unexpectedly, only the monoacyl group was cleaved to produce sn-1 LPIPs or sn-2 LPIPs. Alternatively, it was found that, when the methylamine concentration was 2.13%, unexpectedly, only the monoacyl group was cleaved under reaction conditions of 120 minutes to produce LPIP.

As used herein, "deacylation" refers to the cleavage (removal) of the acyl group by cleaving the fatty acid bond at the 1st and/or 2nd positions of phosphatidylinositol phosphate (which may be monophosphate, diphosphate, or triphosphate; also known as PIPs). Unless otherwise specified herein, "deacylation" may include both "demonoacylation," in which one acyl group is cleaved (removed), and "dediacylation," in which two acyl groups are cleaved (removed). As generally used herein, it includes "demonoacylation." "Demonoacylation" may be either at the sn-1 position or at the sn-2 position.

In one specific embodiment, methylamine/H ₂ O/methanol/n-butanol=1.92/38.08/40/10 (deacylation reaction solution) is added to the dried PIPs, and incubated at 53° C. for 5 minutes. The reaction is stopped by adding ice-cooled deacylation reaction solution (Figure 1 shows an exemplary result of measuring the product after leaving it on ice for 1 minute and then drying it). One or more (or all) of various conditions such as the solvent composition, reaction temperature, reaction time, and catalyst (typically methylamine) concentration can be appropriately changed as long as demonoacylation occurs. In a preferred embodiment, it is understood that the object of the present invention can be achieved by setting the amount of methylamine to 1 to 3%.

The starting materials used in the production method of the present invention, phosphatidylinositol monophosphate, phosphatidylinositol diphosphate, or phosphatidylinositol triphosphate, can be produced by methods known in the art or commercially available products. For example, any type of PIP can be produced by the method described in Stuart J. Conway et al., Org. Biomol. Chem., 2010, 8, 66-76.

In addition, a method for easily separating a lyso form having a desired acyl group at a desired position by preparing different raw materials is also provided. In another aspect, the present invention provides a method for easily separating a lyso form having a desired acyl group at a desired position (position 1 or position 2) by (A) providing a phosphatidylinositol monophosphate, phosphatidylinositol diphosphate, or phosphatidylinositol triphosphate having acyl groups of different masses at positions 1 and 2, in which an acyl group having a desired mass is present at a desired position (position 1 or 2); (B) contacting the phosphatidylinositol monophosphate, phosphatidylinositol diphosphate, or phosphatidylinositol triphosphate with an alkylamine to deacylate the phosphatidylinositol monophosphate, phosphatidylinositol diphosphate, or phosphatidylinositol triphosphate. and (C) a step of isolating lysophosphatidylinositol monophosphate, lysophosphatidylinositol diphosphate, or lysophosphatidylinositol triphosphate having a desired acyl group at a desired position (a method for producing them separately), the method comprising the steps of (a) removing lysophosphatidylinositol monophosphate, lysophosphatidylinositol diphosphate, or lysophosphatidylinositol triphosphate having a desired acyl group at a desired position, the step comprising (a) removing lysophosphatidylinositol monophosphate, lysophosphatidylinositol diphosphate, or lysophosphatidylinositol triphosphate having a desired mass of acyl group.

In the method of the present invention, phosphatidylinositol monophosphate, phosphatidylinositol diphosphate, or phosphatidylinositol triphosphate having acyl groups of different masses at the 1st and 2nd positions can be produced with reference to Stuart J. Conway et al., Org. Biomol. Chem., 2010, 8, 66-76, and will be described in detail below. The above-mentioned conditions can be appropriately applied for deacylation. Any means that can separate the desired lysophosphatidylinositol monophosphate, lysophosphatidylinositol diphosphate, or lysophosphatidylinositol triphosphate based on differences in mass, hydrophobicity, etc. can be used for extraction. For example, chromatography can be used. Of course, if the product can be used without purification, the purification step is not essential.

The following is a general synthesis scheme for starting materials (such as PIPs) having the desired fatty acid and phosphate groups. Examples of the synthesis of starting materials (such as PIPs) are comprehensively described in Stuart J. Conway et al., Org. Biomol. Chem., 2010, 8, 66-76, and they can be synthesized from phosphoramidite precursors (diacylglycerol phosphate derivatives) and the like.

(General example of phosphoramidite precursor synthesis)

(Here, R ¹ and R ² are each independently any fatty acid residue, R ^B and R ^C are each independently hydrogen or an alkyl group (e.g., an alkyl group having 1 to 12 carbon atoms, such as methyl and ethyl), and PG ^A , PG ^P1 and PG ^P2 are each independently a protecting group. Examples of the protecting group include, but are not limited to, a benzyl group, a p-methoxybenzyl group, a tert-butyl group and the like. When a benzyl group is used, hydrogenation is performed during elimination, so that it is preferable not to use it when introducing a fatty acid residue containing a double bond.)
Compound P-1 (e.g., (+)-1-O-(4-methoxybenzyl)-2-O-methoxymethyl-glycerol) is treated with an carboxylic acid halide under appropriate conditions (e.g., adding dimethylaminopyridine (DMAP) and acetyl chloride to a dichloromethane solution of P-1 at 0° C. and stirring at room temperature) to obtain compound P-2. Compound P-2 is treated with an appropriate condition (e.g., using trifluoroacetic acid) to deprotect the protecting group PG ^P1 (e.g., methoxymethyl group) of the hydroxy group to obtain compound P-3. Compound P-3 is treated with an carboxylic acid halide under appropriate conditions (e.g., adding dimethylaminopyridine (DMAP) and octadecanoyl chloride to a dichloromethane solution of compound P-3 at 0° C. and stirring at room temperature) to obtain compound P-4. Compound P-4 is subjected to an appropriate condition (e.g., using 2,3-dichloro-5,6-dicyano-p-benzoquinone (DDQ) or ammonium hexanitracerate(IV) (CAN)) to deprotect the protecting group PG ^P2 (e.g., 4-methoxybenzyl group) of the hydroxy group to obtain compound P-5. Compound P-5 is subjected to an appropriate condition with an aminophosphine (e.g., (4-methoxybenzyloxy)bis(N,N-diisopropylamino)phosphine) (e.g., by adding 1H-tetrazole to a dichloromethane solution at room temperature and stirring), to obtain phosphoramidite P-6.

(General manufacturing example of inositol precursor)

(PG ¹ , PG ^A , and PG ² are each independently a protecting group. Examples of the protecting group include, but are not limited to, methoxybenzyl.)
Compound I-1 is subjected to appropriate conditions (for example, 4-methoxybenzyl chloride and sodium hydride are added to a DMF solution of compound I-1 at 0° C., followed by stirring at room temperature) to obtain compound I-2 having a protecting group PG ^A (for example, a 4-methoxybenzyl group) introduced therein. Compound I-2 is subjected to appropriate conditions (for example, chloromethyl methyl ether and sodium hydride are added to a DMF solution of compound I-2 at 0° C., followed by stirring at room temperature) to obtain compound I-3 having a protecting group PG ¹ (for example, a methoxymethyl group) introduced therein. Compound I-3 is subjected to appropriate conditions (for example, diisobutylaluminum hydride (DIBAL) is added to a dichloromethane/hexane solution of compound I-3 at 0° C., followed by stirring at room temperature) to obtain compound I-4. Compound I-4 is subjected to an appropriate condition (for example, 4-methoxybenzyl chloride and sodium hydride are added to a DMF solution of compound I-4 at 0° C., followed by stirring at room temperature) to obtain compound I-5 having a protecting group PG ² (for example, a 4-methoxybenzyl group). Compound I-5 is subjected to an appropriate condition (for example, hydrochloric acid is added to a methanol solution of compound I-5, followed by reflux) to obtain compound I-6. Compound I-6 is subjected to an appropriate condition (for example, toluenesulfonic acid monohydrate is added to a dichloromethane solution of compound I-6, followed by reflux).

and reflux, then perform optical resolution, and then add toluenesulfonic acid monohydrate and

and react to obtain compound I-7 (e.g.

Compound I-7 is subjected to appropriate conditions to obtain compound I-8 (l, m, and n are integers from 0 to 6, and the sum of l, m, and n is 6), which has one hydroxy group, m hydroxy groups protected by protecting group PG ¹ , and n hydroxy groups protected by PG ² .

(General example of phosphatidylinositol production)

(In the formula, l, m, and n are integers from 0 to 6, and the sum of l, m, and n is 6.)
An appropriate phosphorus reagent (e.g., bis(4-methoxybenzyloxy)(N,N-diisopropylamino)phosphine) is used on the hydroxyl group of an inositol precursor PI-1 (e.g., 1D-2,3,4,5,6-penta-O-(4'-methoxybenzyl)-1-O-(methoxymethyl)-myo-inositol) under appropriate conditions (e.g., adding 1H-tetrazole to a dichloromethane solution of PI-1 and stirring at room temperature for 10 to 14 hours) to obtain PI-2 (e.g., 1D-2,3,4,5,6-penta-O-(4'-methoxybenzyl)-1-O-(methoxymethyl)-myo-inositol-4-(bis(4-methoxybenzyloxy)phosphate)) modified with a phosphate group. PI-2 is subjected to appropriate conditions (e.g., using trifluoroacetic acid) to remove the protecting group PG ¹ (e.g., methoxymethyl group) to give PI-3 (e.g., 1D-2,3,4,5,6-penta-O-(4′-methoxybenzyl)-myo-inositol-4-(bis(4-methoxybenzyloxy)phosphate)). PI-3 and a phosphoramidite precursor (e.g., (1-acetyloxy-2'-octadecanoyloxypropyl)(4-methoxybenzyl)diisopropylphosphoramidite) are subjected to appropriate conditions (e.g., 1H-tetrazole and (1-acetyloxy-2-octadecanoyloxypropyl)(4-methoxybenzyl)diisopropylphosphoramidite are added to a dichloromethane solution of PI-3, followed by stirring at room temperature for 10 to 14 hours, followed by addition of metachloroperbenzoic acid (mCPBA) at -78°C, and stirring at room temperature for 20 minutes) to obtain PI-4. The protecting group PG ² (e.g., 4-benzyl group) of PI-4 is deprotected under appropriate conditions (e.g., adding ammonium hexanitracerate(IV) (CAN) to an acetonitrile solution of PI-4 and stirring at room temperature for 45 minutes) to obtain PI-5 (e.g., 1D-myo-inositol-1-(1'-O-acetyl-2'-O-octadecanoyl-sn-glycer-3-yl phosphate)).

The phosphatidylinositol phosphate produced by the above-mentioned production method has, for example, the following formula:

These include, but are not limited to, those shown in the table.

(General production example of Lyso-phosphatidylinositol phosphate)

A solution of phosphatidylinositol 1 (e.g., 1D-myo-inositol-1-(1'-O-acetyl-2'-O-octadecanoyl-sn-glycer-3-yl phosphate) (e.g., a chloroform/methanol (9/1) solution can be used, but the ratio of the solvents for preparing the solution can be changed as appropriate, and other solvents can be used as long as the reaction is promoted.) is dried with an inert gas such as N2, and then a suitable reagent (e.g., a methylamine solution (exemplary solvents include, but are not limited to, methylamine/water/methanol/1-butanol (4.8/35.2/40/10). The ratio of the solvent and methylamine ... added. A different solvent can also be used.) is added at an appropriate temperature (e.g., 53°C), stirred for an appropriate time (e.g., 5 minutes can be exemplified, but longer or shorter times are acceptable as long as demonoacylation is achieved. For example, it has been observed that demonoacylation occurs even for 1 minute or 30 seconds.) at an appropriate temperature (e.g., 53°C can be exemplified, but higher or lower times are acceptable as long as demonoacylation is achieved.) to quench the reaction (e.g., by cooling on ice), and then appropriately post-treatment is performed to obtain the desired lyso-phosphatidylinositol. Here, even if the elimination reaction of the acyl group does not proceed regioselectively, when the acyl groups COR ¹ and COR ² introduced into phosphatidylinositol 1 are different (e.g., when COR ¹ is an acetyl group and COR ² is an octadecanoyl group), compounds 2 and 3 from which only one acyl group has been eliminated have different molecular weights, and therefore it is possible to separate and purify each compound.

Detection method
In another aspect, the present invention provides a method for detecting, identifying or quantifying lysophosphatidylinositol phosphate, comprising the steps of: (A) concentrating phospholipids by contacting a sample with an anion exchange resin; (B) protecting the phosphate group in the phospholipid with a protecting group; and (C) detecting, identifying or quantifying lysophosphatidylinositol phosphate in the phospholipid by mass spectrometry. Phosphoinositides including lysophosphatidylinositol phosphate have many phosphate groups and are present in trace amounts (in biological lipid extracts), so they are subject to ion suppression by other lipids in the sample, making them the most difficult membrane phospholipids to analyze, which has been considered impossible in the past. However, by using the method of the present invention, it has become possible to detect, identify and quantify lysophosphatidylinositol phosphate. The method of the present invention can set the lower limit of quantification to several hundred amol to several fmol, and can measure many types of LPIPs whose existence in the living body was unknown. Thus, the present invention provides a mass spectrometry-based method capable of absolute quantification in a variety of biological trace samples.

Conventional measurement techniques have made it possible to measure PIPs, but the present invention has found that lysophosphatidylinositol phosphates can be identified and detected. Therefore, the present invention provides, for the first time in history, a technique for identifying and detecting lysophosphatidylinositol phosphates.

As the protecting group for the phosphate group of the present invention, any protecting group can be used as long as it can provide protection, but preferably, it is one that does not interfere with mass spectrometry or can be removed before mass spectrometry, such as an alkyl group (e.g., a methyl group). Preferably, the protecting group contains an alkyl group, and more preferably, it contains a methyl group.

The mass spectrometry used in the present invention can be any technique, but preferably involves selected reaction monitoring (SRM) using a triple quadrupole mass spectrometer.

The SRM used in the present invention further includes a step of detecting, identifying or quantifying the fatty acid side chains and/or phosphate groups of the lysophosphatidylinositol phosphate using reverse phase column chromatography.

Preferably, in the reverse phase column chromatography, diacylglycerol is selected as a product ion (fragment ion) to detect, identify or quantify the fatty acid side chains and/or phosphate groups.

Specific examples of phosphoinositide measurement techniques are given below.

After examining this method, it was found that LPIPs can also be measured.

In one aspect, PIP1 and PIP2 are quantified as the sum of three isomers (structural isomers with different positions of phosphorylated hydroxyl groups). The procedure is outlined below.

1 High recovery of phosphoinositides such as lysophosphatidylinositol phosphates in vivo The total lipid fraction obtained by the Bligh-Dyer method is further fractionated using an anion exchange column (DEAE cellulose column). Phospholipids are gradually separated and eluted due to the difference in the ion exchange capacity of the polar parts of each phospholipid, and a fraction rich in phosphoinositides can be recovered in high amounts, and lysophosphatidylinositol phosphates can also be recovered in high amounts.

2. Stabilization of the molecule Phosphoinositides, which have many phosphate groups on the inositol ring, are unstable and tend to be adsorbed by many materials, including metals, and it is believed that lysophosphatidylinositol phosphates also have similar properties. Therefore, phosphoinositides are rapidly lost in the analysis vial during the waiting time from recovery to measurement, and tailing is observed in the detected elution peak. After recovering phosphoinositides at a high level, the inventors prepared stable derivatives that suppressed decomposition and adsorption by methylating the phosphate groups, and performed robust analysis. This method can also be applied to lysophosphatidylinositol phosphates.

3 Highly sensitive analytical method For the measurement of phosphoinositides and lysophosphatidylinositol phosphates, a selective reaction monitoring method (SRM) using a triple quadrupole mass spectrometer is used, which is a highly sensitive and quantitative analytical method. Phosphoinositides are easily detected as negative ions due to their structure, but in the analytical method of the present invention, the phosphate groups are protected by methylation or other methods, making them easier to detect as positive ions. Lysophosphatidylinositol phosphates also show similar properties. Diacylglycerol (DG) is selected as a characteristic fragment ion for each molecular species. Analysis is performed by separating and eluting each molecular species using a reversed-phase column (C8) by utilizing the difference in hydrophobicity of the fatty acid side chains. Phosphoinositides of the same molecular species are eluted in the order of PIP3, PIP2, and PIP1 due to the difference in the number of phosphate groups, and this difference in elution time is utilized for analysis. Quantitative analysis is carried out by converting the peak area of the chromatogram of each molecular species smoothed by the Gaussian method into a numerical value, and applying it to a calibration curve prepared using various 17:0/20:4 synthetic products that are hardly present in the body to calculate. This analysis method of the present invention is highly sensitive and excellent in quantitative properties, and can be used to analyze human clinical specimens and samples derived from animal tissues in addition to cultured cells. The same analysis is also possible for lysophosphatidylinositol phosphates, and since they are eluted in the order of LPIP3, LPIP2, and LPIP1, as well as the difference in molecular weight, the difference in elution time is utilized for analysis, and the peak area of the chromatogram of each molecular species smoothed by the Gaussian method is converted into a numerical value, and compared with a standard product, and can be calculated by applying it to a calibration curve prepared.

The present invention provides a technology for detecting lysophosphatidylinositol phosphates, which can also be used as disease markers, by distinguishing them from other substances, which can also be used to diagnose cancer.

Metabolic abnormalities of diacyl inositol phospholipids (PIPs), which are considered to be precursors of LPIPs, are known to be related to various pathologies, and are useful as therapeutic targets and diagnostic markers. Therefore, the LPIPs of the present invention, which are structurally closely related to diacyl inositol phospholipids (PIPs), are also known to be related to various pathologies, and are expected to be useful as therapeutic targets and diagnostic markers. Specifically, the following uses are expected. In addition, as explained in Biochemistry, Vol. 83, No. 6, pp. 525-535, 201, it is known that lysophosphatidylinositol as a lipid mediator has various activities, including G protein receptors such as GPR55 and its receptor. Therefore, the compounds of the present invention in which lysophosphatidylinositol is phosphorylated in various forms are expected to be directly or indirectly related to various physiological uses in relation to lysophosphatidylinositol and its receptors. In addition, as described in Fujii et al. Folia Pharmacol Jpn. Vol. 142, 2013, pp. 236-240, inositol polyphosphates generated from PIPs by the action of phospholipase C control calcium dynamics in cells, and abnormalities in calcium dynamics are related to a wide range of pathologies including psychiatric disorders, muscular disorders, and cardiac disorders. Therefore, the LPIPs of the present invention, which are structurally closely related to the inositol polyphosphates, are also expected to be useful as therapeutic targets and diagnostic markers.

Measurement, Detection and Identification of Phosphatidylinositol Phosphates and Lysophosphatidylinositol Phosphates
In one aspect, the present invention provides a method for measuring, detecting or identifying phosphatidylinositol phosphate and/or lysophosphatidylinositol phosphate in a sample. The method includes the steps of A) subjecting the sample to mass spectrometry (MS); and B) identifying the position of the phosphate group of the phosphatidylinositol phosphate and/or lysophosphatidylinositol phosphate by the elution position of the peak in the MS. In one embodiment, the sample is further subjected to chromatography. In one embodiment, the sample is further subjected to ion mobility separation (IMS).

In one embodiment, the position of the phosphate group of the phosphatidylinositol phosphate and/or lysophosphatidylinositol phosphate of the present invention can be identified by comparison with a standard. Alternatively, the elution position and elution time of the standard can be calculated and set in advance, and then the identification can be performed by comparison with the calculated elution position and elution time. In addition, as a method for identifying PIPs and LPIPs, for example, a method of adding stable isotopes of phosphate or inositol ( ^13C or deuterium) to a sample in advance to examine whether they can be replaced with natural isotopes can be mentioned.

In another aspect, the present invention provides a method for measuring, detecting, or identifying phosphatidylinositol phosphate and/or lysophosphatidylinositol phosphate in a sample. The method includes the steps of A) subjecting the sample to ion mobility separation-mass spectrometry (IMS-MS); and B) identifying the positions of the phosphate groups of the phosphatidylinositol phosphate and/or lysophosphatidylinositol phosphate based on the elution positions of peaks in the IMS-MS.

In one embodiment, the present invention is characterized in that the presence or absence of acyl groups contained in the phosphatidylinositol phosphate and/or lysophosphatidylinositol phosphate is also identified in the identification step. Some phospholipids in a sample do not have acyl groups, but since they are not deacylated in the measurement of the present invention, they can be measured in an intact state, and therefore the presence or absence of acyl groups can be identified.

In one embodiment, the total molecular weight of the acyl groups contained in the phosphatidylinositol phosphate and/or lysophosphatidylinositol phosphate is also determined in the determination step of the present invention. In the measurement of the present invention, the molecular weight is determined by mass spectrometry. Therefore, once it is determined that it is phosphatidylinositol phosphate and/or lysophosphatidylinositol phosphate, the total molecular weight of the bound acyl groups is determined from the molecular weight.

In a preferred embodiment, the type of acyl group contained in the phosphatidylinositol phosphate and/or lysophosphatidylinositol phosphate is also identified in the identification step of the present invention. In the measurement of the present invention, the molecular weight is identified by mass spectrometry. Therefore, once it is identified as phosphatidylinositol phosphate and/or lysophosphatidylinositol phosphate, the total molecular weight of the acyl groups bound to it is identified from the molecular weight, and in the case of phosphatidylinositol phosphate, the monoacyl form is identified by mass spectrometry, so the molecular weight of one of the acyl groups is identified. Once the molecular weight of one is identified, the molecular weight of the other is also identified, and the type of acyl group is identified from the molecular weight. A method for identifying the type of acyl group from the molecular weight based on the results of measurement by MS and MS/MS is described, for example, in Clark J et al., Nat Methods. 2011 Mar;8(3):267-72. Please refer to doi:10.1038/nmeth.

In one embodiment, the measurement, detection, or identification in the present invention is characterized in that phosphatidylinositol phosphate and/or lysophosphatidylinositol phosphate are distinguished from other inositol-containing phospholipids. This is possible because the measurement, detection, or identification method of the present invention allows measurements to be performed while retaining the acyl groups. More specifically, phosphatidyl phosphate, lysophosphatidylinositol phosphate, and inositol-containing phosphoric acid such as glycerophosphoinositol can be distinguished by the difference in the molecular weight of the acyl groups by performing measurements that retain the acyl groups.

In one embodiment, the sample to be measured in the present invention is prepared under conditions in which decomposition of phosphatidylinositol phosphate and/or lysophosphatidylinositol phosphate does not occur.

Therefore, in the measurement, detection or identification of the present invention, the conditions under which phosphatidylinositol phosphate and/or lysophosphatidylinositol phosphate are not decomposed can include any conditions under which decomposition is not performed, particularly including not performing a deacylation treatment, and other conditions such as not performing a treatment that denatures the sample and performing a treatment that introduces a protecting group into the phosphate group.

The liquid column chromatography used in the present invention can be a chiral column, reverse phase column, normal phase column, ion exchange column, etc., and any column chromatography that separates hydrophilic and hydrophobic groups can be used.

When used in the present invention, chromatography (gas chromatography or liquid column chromatography) can be combined with ion mobility separation. Without wishing to be bound by theory, the present invention has shown that ion mobility separation can more clearly separate the positions of phosphate groups of phosphoinositides and/or lysophosphoinositides, and by combining any chromatography (e.g., liquid column chromatography) with ion mobility separation and further performing mass spectrometry, the positions of phosphate groups of phosphoinositides and/or lysophosphoinositides can be separated and measured, detected or identified while retaining the acyl groups. Alternatively, even if ion mobility separation is not used, the positions of phosphate groups of phosphoinositides can be separated and measured, detected or identified while retaining the acyl groups by using mass spectrometry (MS), and further, the positions of phosphate groups can be separated and measured, detected or identified in combination with chromatography such as liquid chromatography using a chiral column. Alternatively, when ion mobility separation is used, it may be combined with mass spectrometry as it is without using chromatography.

In a preferred embodiment of the present invention, the method further comprises a step of quantifying phosphatidylinositol phosphate and/or lysophosphatidylinositol phosphate.

In one embodiment, the sample used in the present invention is an intact sample. Here, the term "intact sample" includes conditions under which the acyl groups of phosphoinositides and lysophosphoinositides are maintained, including, but not limited to, conditions under which the sample has not been denatured by heating, oxidized by the action of oxygen in the air, moisture, heat, light, metal ions, microorganisms or enzymes, hydrolysis in an aqueous solvent, or deacylation in the presence of an alkylamine such as methylamine.

In one embodiment, in the present invention, the position of the phosphate group of phosphatidylinositol phosphate and/or lysophosphatidylinositol phosphate is identified without labeling the sample. Since there is no need to label with a fluorescent label or a radioisotope, the sample in the living body can be analyzed under the same conditions as the living body or under conditions similar to those conditions, and an analysis that reflects the information of the living body can be performed.

(Measuring, detecting or identifying devices)
In one aspect, the present invention provides an apparatus for measuring, detecting or identifying the positions of acyl and phosphate groups of phosphatidylinositol phosphate and/or lysophosphatidylinositol phosphate in a sample. Here, the apparatus includes a mass spectrometer, and preferably further includes a chromatography apparatus or an ion mobility separation apparatus, for example, the chromatography apparatus may include at least one apparatus for gas chromatography and liquid chromatography. Without wishing to be bound by theory, since phosphatidylinositol phosphate and/or lysophosphatidylinositol phosphate can be measured using liquid column chromatography and a mass spectrometer in the present specification, it is understood that they can be separated and measured in the same way by gas chromatography. When separation between phosphate positional isomers is more accurately performed, it is preferable to combine with a chromatography apparatus or an ion mobility separation apparatus. As a specific embodiment, the present invention includes a mass spectrometer (MS) apparatus and an application unit that applies the sample to the MS under conditions that do not decompose phosphatidylinositol phosphate. In a more specific embodiment, the present invention includes a liquid column chromatography-mass spectrometry (LC-MS) device and an application section for applying the sample to the LC-MS under conditions that do not decompose phosphatidylinositol phosphates.

In another aspect, the present invention provides an apparatus for measuring, detecting, or identifying the positions of acyl and phosphate groups of phosphatidylinositol phosphate and/or lysophosphatidylinositol phosphate in a sample, the apparatus comprising an ion mobility separation-mass spectrometry (IMS-MS) device and an application section for applying the sample to the IMS-MS under conditions that do not decompose the phosphatidylinositol phosphate and/or lysophosphatidylinositol phosphate.

In the present invention, when LC-MS is used, it may include a means for performing ion mobility separation, if necessary.

The liquid column chromatography used in this specification is selected from the group consisting of chiral columns, reverse phase columns, normal phase columns, ion exchange columns, and columns that separate hydrophilic and hydrophobic groups.

In one embodiment, the method further includes a means for detecting or quantifying the phosphatidylinositol phosphate and/or lysophosphatidylinositol phosphate. Examples of such a means for detecting or quantifying the phosphatidylinositol phosphate include a method for calculating and quantifying the detected peak obtained by comparison with an internal standard.

(Separation, purification and pure production of phosphoinositides and/or lysophosphoinositides)
In one aspect, the present invention provides a method for separating or purifying phosphatidylinositol phosphates and/or lysophosphatidylinositol phosphates in a sample according to the position of the phosphate group while retaining the acyl group. The method includes the steps of A) subjecting the sample to mass spectrometry (MS); and B) collecting phosphatidylinositol phosphates and/or lysophosphatidylinositol phosphates having a phosphate group at a position of interest specified by the elution position of the peak from the MS eluate. In one embodiment, the sample is further subjected to chromatography or ion mobility separation (IMS).

In one aspect, the present invention provides a method for producing a sample containing phosphatidylinositol phosphates and/or lysophosphatidylinositol phosphates having phosphate groups at positions of less types (e.g., one type) than the number of types of phosphate group positions of phosphatidylinositol phosphates and/or lysophosphatidylinositol phosphates contained in a mixture in which two or more phosphate group positions of phosphatidylinositol phosphates and/or lysophosphatidylinositol phosphates are present. This method includes the steps of A) applying the sample to chromatography, ion mobility separation (IMS) or mass spectrometry (MS); and B) collecting a fraction containing phosphatidylinositol phosphates and/or lysophosphatidylinositol phosphates having phosphate groups at a position of interest specified by the elution position of a peak from the eluate of the chromatography, IMS or MS. In one embodiment, the sample is subjected to any combination of chromatography, ion mobility separation and mass spectrometry.

In the present invention, the technique for collecting phosphatidylinositol phosphates and/or lysophosphatidylinositol phosphates having a phosphate group at a desired position identified by the elution position of the peak can be any technique known in the art, for example, an appropriate instrument such as an autosampler can be used, or the samples can be collected manually.

Here, examples of mixtures in which phosphatidylinositol phosphate and/or lysophosphatidylinositol phosphate are present at two or more positions of the phosphate group include biological samples, foods, metabolites, and any other samples that contain or are assumed to contain phosphoinositides and/or lysophosphatidylinositol phosphate. In particular, in the present invention, the mixture may contain two or three isomers of the three isomers of monophosphate phosphatidylinositol (phosphatidylinositol-3-monophosphate, phosphatidylinositol-4-monophosphate, phosphatidylinositol-5-monophosphate), and may similarly contain two or three isomers of the three isomers of monophosphate lysophosphatidylinositol, and may contain two or three isomers of diphosphate phosphatidylinositol (phosphatidylinositol-3,4-diphosphate, phosphatidylinositol-3,5-diphosphate, phosphatidylinositol-5-monophosphate). The mixture may contain two or three isomers of the three isomers of diphosphate lysophosphatidylinositol (inositol-4,5-diphosphate), and similarly may contain two or three isomers of the three isomers of diphosphate lysophosphatidylinositol, which may be separated to produce a sample containing phosphatidylinositol phosphates and/or lysophosphatidylinositol phosphates having phosphate groups at positions that are fewer in number (e.g., one type) than the number of types of phosphate group positions of the phosphatidylinositol phosphates and/or lysophosphatidylinositol phosphates contained in the mixture. In the present specification, phosphatidylinositol phosphate having a phosphate group at one position and lysophosphatidylinositol phosphate having a phosphate group at one position may be any isomer having seven types of binding patterns of phosphate groups, which are phosphatidylinositol-3-monophosphate, phosphatidylinositol-4-monophosphate, phosphatidylinositol-5-monophosphate, phosphatidylinositol-3,4-diphosphate, phosphatidylinositol-3,5-diphosphate, phosphatidylinositol-4,5-diphosphate, phosphatidylinositol-3,4,5-triphosphate, and lyso forms thereof. The present invention also covers a technique capable of separating a part of the mixture from the mixture.

In this specification, "types less than the number of types of phosphate group positions of phosphatidylinositol phosphate and/or lysophosphatidylinositol phosphate contained in the mixture" means that, when the number of types of phosphate group positions of phosphatidylinositol phosphate and/or lysophosphatidylinositol phosphate contained in the mixture is n-1 or less. Preferably, the number of types less than the number of types of phosphate group positions of phosphatidylinositol phosphate and/or lysophosphatidylinositol phosphate contained in the mixture is 1, but is not limited thereto and may be 1 type, 2 types, 3 types, 4 types, etc. In particular, it may be advantageous to be able to separate the three isomers of phosphatidylinositol in the monophosphate form (phosphatidylinositol-3-monophosphate, phosphatidylinositol-4-monophosphate, phosphatidylinositol-5-monophosphate), the three isomers of lysophosphatidylinositol in the monophosphate form, the three isomers of phosphatidylinositol in the diphosphate form (phosphatidylinositol-3,4-diphosphate, phosphatidylinositol-3,5-diphosphate, phosphatidylinositol-4,5-diphosphate), or the three isomers of lysophosphatidylinositol in the diphosphate form.

(General Technology)
The molecular biological, biochemical and microbiological techniques used herein are well known and commonly used in the art, and are described, for example, in Sambrook J. et al. (1989). Molecular Cloning: A Laboratory Manual, Cold Spring Harbor and its 3rd Ed. (2001); Ausubel, FM (1987). Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Ausubel, FM (1989). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Innis, MA (1990). PCR Protocols: A Guide to Methods and Applications, Academic Press; Ausubel, FM (1992). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates; Ausubel, FM (1995). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates; Innis, MA et al.(1995). PCR Strategies, Academic Press; Ausubel, FM(1999). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Wiley, and annual updates; Sninsky, JJ et al.(1999). PCR Applications: Protocols for Functional Genomics, Academic Press, Special Edition of Experimental Medicine "Gene Introduction & Expression Analysis Experimental Methods" Yodosha, 1997, etc., and the relevant parts (which may be the entirety) of these are incorporated by reference in this specification.

For the DNA synthesis techniques and nucleic acid chemistry for producing artificially synthesized genes, see, for example, Gait, M.J.(1985). Oligonucleotide Synthesis: A Practical Approach, IRL Press; Gait, M.J.(1990). Oligonucleotide Synthesis: A Practical Approach, IRL Press; Eckstein, F.(1991). Oligonucleotides and Analogues: A Practical Approach, IRL Press; Adams, R.L. et al.(1992). The Biochemistry of the Nucleic Acids, Chapman &H all; Shabarova, Z. et al.(1994).Advanced Organic Chemistry of Nucleic Acids, Weinheim; Blackburn, G.M. et al.(1996). Nucleic Acids in Chemistry and Biology, Oxford University Press; Hermanson, G.T.(I996). Bioconjugate Techniques, Academic Press, et al., which are incorporated herein by reference in their relevant portions.

In this specification, "or" is used when "at least one or more" of the items listed in the sentence can be adopted. The same applies to "alternative." When it is stated in this specification that "within the range of" two values, the range includes the two values themselves.

All references cited herein, including scientific literature, patents, patent applications, and the like, are hereby incorporated by reference in their entirety to the same extent as if each was specifically set forth herein.

The present invention has been described above by showing preferred embodiments for ease of understanding. The present invention will be described below based on examples, but the above description and the following examples are provided for illustrative purposes only and are not provided for the purpose of limiting the present invention. Therefore, the scope of the present invention is not limited to the embodiments or examples specifically described in this specification, but is limited only by the scope of the claims.

Examples are described below. Little research has been done focusing on the diversity (molecular species) of fatty acid chains bound to the sn-1,2 hydroxyl groups of glycerol, which is one of the structural characteristics of phosphoinositides. The mainstream method for measuring intracellular phosphoinositide levels is the HPLC method, which analyzes deacylated phosphoinositides labeled with radioisotopes. However, this semi-quantitative analysis method is limited in the samples it can be used for (difficult in vivo), and above all, it cannot measure at the molecular species level. Therefore, the inventors have established a new analytical method with excellent quantitative properties using LC-MS/MS. This analytical method is capable of analyzing phosphoinositides at the molecular species level for a wide range of biological samples.

Where necessary, all animal experiments (preparation of biological samples, etc.) used in the following examples were carried out in accordance with the Akita University Guidelines for Animal Experiments. Furthermore, experiments involving human subjects were carried out in accordance with the Declaration of Helsinki. Specific reagents used were those listed in the examples, but equivalent products from other manufacturers (Sigma-Aldrich, Wako Pure Chemical Industries, Nakarai, R&D Systems, USCN Life Science INC, etc.) can also be used.

(Abbreviation)
In addition to the abbreviations explained in this specification, the following abbreviations are also used.

PI: phosphatidylinositol PI3P or PI(3)P: phosphatidylinositol-3-monophosphate PI4P or PI(4)P: phosphatidylinositol-4-monophosphate PI5P or PI(5)P: phosphatidylinositol-5-monophosphate (PI3P, PI4P, or PI5P are referred to as PIP1)
PI(3,4) _P2 : phosphatidylinositol-3,4-bisphosphate PI(3,5) _P2 : phosphatidylinositol-3,5-bisphosphate PI(4,5) _P2 : phosphatidylinositol-4,5-bisphosphate (PI(3,4) _P2 or PI(3,5) _P2 or PI(4,5) _P2 are abbreviated as PIP2)
PI(3,4,5) _P3 : phosphatidylinositol-3,4,5-triphosphate (PI(3,4,5) _P3 is abbreviated as PIP3)
PIP1, PIP2, and PIP3 are collectively referred to as PIPs.

Structures in which PIPs are bound to fatty acids or hydrocarbons via only either the sn-1 hydroxyl group or the sn-2 hydroxyl group of glycerol are collectively referred to as LPIPs.

Example 1: Production and identification of lysophosphatidylinositol phosphates
In this example, lysophosphatidylinositol phosphates were produced, and a new method for identifying these novel substances was also developed.

(Chemical Substances)
The following samples were used as standards and starting materials:

(material and method)
(Materials used)
Phosphatidylinositol phosphates 17:0/20:4-PI3P, 17:0/20:4-PI4P, 17:0/20:4-PI5P, 17:0/20:4-PI(3,4)P2, 17:0/20:4-PI(3,5) _P2 , 17:0/20:4-PI(4,5) _{P2 ,} 17:0/20:4-PI(3,4,5) _P3 , and other synthetic glycerophospholipid standards used as starting materials were purchased from Avanti Polar Lipids (Alabaster, AL, USA). Trimethylsilyldiazomethane (TMS-diazomethane) was purchased from Tokyo Kasei. Ultrapure water was obtained from Kanto Chemicals (Tokyo, Japan). All other solvents were of HPLC or LC-MS grade, and other chemical reagents were of analytical grade. They were obtained from Wako Pure Chemicals.

(Synthesis Method)
Phosphatidylinositol phosphates (monophosphate, diphosphate, and triphosphate) were used as starting materials and deacylated by O→N transacylation with methylamine. The following conditions were set for deacylation so that only one acylation was possible:

(Experimental conditions)
100 pmol of 1-heptadecanoyl-2-arachidonoyl-sn-glycero-3-phospho-1'-myo-inositol-4'-monophosphate (17:0/20:4-PI4P) dissolved in chloroform/methanol (1/1) was The reaction mixture was dried over _N2 gas, and 0.3 mL of 2.13% methylamine solution (methylamine/water/methanol/1-butanol (1.92/38.08/40/10)) or 10.7% methylamine solution (methylamine/water/methanol/1-butanol (9.6/30.4/40/10)) was added at 53°C or 37°C. After stirring, the mixture was left to stand at 53°C or 37°C for 0, 5, 10, 30, 60, or 120 minutes, and the reaction was quenched by adding ice-cold methanol solution. The reaction mixture was dried over _N2 gas, and the desired lysophosphatidylinositol monophosphate (LPIP1: yield about 0-40%) was subjected to methylation protection of the phosphate group and measured by the following method using a triple quadrupole mass spectrometer.

(Measurement and Detection)
"Measurement and detection method by SRM using a triple quadrupole mass spectrometer" is described below.

Methylation reaction of LPIPs phosphate group: For high-sensitivity measurement with a mass spectrometer, a methylation protection reaction of LPIPs phosphate group was performed. A sample containing LPIPs was dissolved in 800 μL of chloroform/methanol (1/1), and 150 μL of trimethylsilyldiazomethane was added and reacted at room temperature for 5 minutes. The reaction was quenched by adding 10 μL of glacial acetic acid. After adding 800 μL of methanol/water/chloroform (48:47:3) and 400 μL of chloroform and stirring, the organic layer was dried with _N2 gas. 27 μL of methanol/70% ethylamine (1:0.13%, v/v) and 9 μL of water were added and stirred.

LC-ESIMS/MS system: LC-ESIMS/MS analysis was performed using a TSQ-Vantage (Thermo-Fisher Scientific) with an UltiMate 3000 LC system (Thermo-Fisher Scientific, Waltham, MA) coupled to an HTC PAL autosampler (CTC Analytics, Zwingen, Switzerland) equipped with a non-metallic injector and a sample loop of 25 μL PEEK tubing. The PIPs fraction was separated by a gradient of mobile phase A (acetonitrile/H ₂ O=4/1, 0.13% ethylamine (70% solution), v/v))/mobile phase B (2-propanol/acetonitrile=4/1, 0.13% ethylamine (70% solution), v/v)) ratio of 70%/30% (0-1 min), 10%/90% over 1-3 min, followed by 10%/90% (3-7.5 min), 70%/30% (7.5-13 min). The flow rate was 220 μL/min, and the chromatography was performed at 60° C. using a GL sciences Inertsil Bio C18 150×1.0 (1.9 μm particle size) column. Typically, 10 μL of sample was injected.

Mass spectrometry conditions: The ion spray voltage was set to 3.0 kV. The heated capillary temperature was set to 270°C. Other parameters were set according to the manufacturer's recommendations. The MS system was tuned using Xcalibur software. LPIPs were measured by the selected reaction monitoring (SRM) method in positive ion mode. The measurement conditions for individual LPIPs in SRM mode are summarized in Table 2. LPIP1, LPIP2, and LPIP3 were identified by the combination of the m/z value of the parent ion (Q1) and the m/z value of the monoacylglycerol measured as the daughter ion (Q2). A schematic diagram is shown in Figure 1-1.

(result)
The horizontal axis shows the reaction time, and the vertical axis shows the amount of 17:0 LPIP1 or 20:4 LPIP1 produced (yield obtained from the peak area ratio of the raw material and the reaction product on the chromatogram). In the conventional method (incubation for 120 minutes with 10.7% methylamine), LPIP1 was not detected, and this was also the case when the reaction time was shortened to 60 minutes. LPIP1 was detected only when the reaction time was further shortened to 5, 10, and 30 minutes. Both 17:0 LPIP1 and 20:4 LPIP1 were produced. A similar reaction time-dependent tendency was observed in the reaction with a methylamine concentration of 2.14%, which was lower than the conventional method. In this case, a small amount of LPIP1 was detected even with reaction times of 60 and 120 minutes, and more LPIP1 was detected with reaction times of 5, 10, and 30 minutes.

As shown in Figure 1-2, the reaction was temperature dependent, and incubation at 37°C for 10 minutes in 10.7% methylamine produced a higher yield of LPIP1 than the reaction at 53°C.

(Discussion)
This method using methylamine has been known to not affect the hydrophilic groups of phospholipids including inositol phospholipids, and does not change the position of the phosphate bonded to inositol, as has been widely used for the purpose of completely deacylating two acyl groups (Serunian LA, et al. Methods Enzymol. 1991;198:78-87, Fujii et al. Folia Pharmacol Jpn. 2013;142:236-240). If the conditions are milder than those, the position and number of phosphate groups are not changed. By adjusting the degree of deacylation by shortening the reaction time, reducing the methylamine concentration, lowering the reaction temperature, or a combination of these, it is possible to deacylate only one acyl, and to synthesize LPIPs corresponding to the diacyl-type PIPs used as the starting material. Next, Table 3 shows examples of LPIPs that can be synthesized in this embodiment using commercially available PIPs synthetic products as raw materials, and Table 4 shows examples in which the position of the phosphate group can be determined. The numbers in the table indicate the number of carbon atoms and the number of double bonds. For those with double bonds, 16:1 = 9-hexadecenoic acid (palmitoleic acid); 18:1 = cis-9-octadecenoic acid (oleic acid); 20:4 = 5,8,11,14-icosatetraenoic acid (arachidonic acid); 22:6 = 4,7,10,13,16,19-docosahexaenoic acid.

Figure 2 (A to G) shows the results of each LPIPs produced from seven types of PIPs starting materials (acyl groups are 17:0 and 20:4) with different phosphorylation patterns. 17:0/20:4-PI3P, 17:0/20:4-PI4P, 17:0/20:4-PI5P, 17:0/20:4-PI(3,4) _P2 , 17:0/20 _: 4-PI(3,5)P2, 17:0/20:4-PI(4,5) _P2 , and 17:0/20:4-PI(3,4,5) _P3 were used as starting materials, and the reaction was quenched by adding ice-cold methanol solution after incubation at 53°C for 10 minutes in the presence of 10.7% methylamine. The reaction mixture was dried with N2 gas, and the phosphate groups of the target LPIPs were protected by methylation and measured using a triple quadrupole mass spectrometer as described above. Regardless of the PIPs used as the raw material, the production of LPIPs was observed in the above-mentioned methylamine reaction.

(Identification of Compounds)
In order to identify the compound of the present invention, accurate mass measurement of specific fragments that support the structure of LPIPs was performed. The "measurement and detection method using a hybrid quadrupole-orbitrap mass spectrometer" is described below. The instrument used was an Orbitrap Q-Exactive Plus, which has a higher mass accuracy than the above-mentioned triple quadrupole mass spectrometer, and the conditions for use are as follows.

LC-ESI MS/MS system: LC-ESI MS/MS analysis was performed using an Orbitrap Q-Exactive Plus (Thermo-Fisher Scientific, Waltham, MA) with an Ultimate 3000 LC system (Thermo-Fisher Scientific, Waltham, MA). The PIPs fraction was separated by a gradient of mobile phase A (acetonitrile/H ₂ O=4/1, 0.13% ethylamine (70% solution), v/v))/mobile phase B (2-propanol/acetonitrile=4/1, 0.13% ethylamine (70% solution), v/v)) ratio of 70%/30% (0-1 min), 10%/90% over 1-3 min, followed by 10%/90% (3-7.5 min), 70%/30% (7.5-13 min). The flow rate was 220 μL/min, and the chromatography was performed at 60° C. using a GL sciences Inertsil Bio C18 150×1.0 (particle size 1.9 μm) column.

Alternatively, the analyses shown in Figures 5C and 5D were performed under the following conditions: PIPs fractions were separated using a gradient of mobile phase A (methanol/H ₂ O/70% ethylamine (20:80:0.13, v/v))/mobile phase B (methanol/H ₂ O/2-propanol/70% ethylamine (5:5:90:0.13, v/v)) with a ratio of 90%/10% (0-0.1 min), 70%/30% over 0.1-3 min, followed by a gradient of 10%/90% (3-15 min). The flow rate was 30 μL/min, and chromatography was performed at 30°C using a Waters X-Bridge C8 (3.5 mm, 1.0 x 150 mm) column.
Typically, 10 μL of sample was injected. Mass spectrometry conditions included ion spray voltage set at 3.0 kV. Heated capillary temperature was set at 250° C. Other parameters were set according to the manufacturer's recommendations. The MS system was tuned using Xcalibur software. LPIPs were measured by selected ion monitoring (SIM) and parallel reaction monitoring (PRM) in positive ion mode.

The LPIPs prepared by incubating the 17:0/20:4-PI4P, 17:0/20:4-PI(4,5) _P2 , and 17:0/20:4-PI(3,4,5) _P3 synthetic products in the presence of 10.7% methylamine at 53°C for 10 minutes as described above were used as samples to confirm that the compounds of the present invention had been synthesized. The theory behind the identification of the specific fragments of LPIP1, LPIP2, and LPIP3 that allowed the identification of the structures of the present invention is as follows.

The identification of LPIP1 will be described with reference to the chromatogram in Figure 3A and the structural formulae of the starting material, product, and fragment in Figure 3B. The results of accurate mass analysis of LPI(4)P and fragments derived from LPI(4)P for two types of LPI(4)P generated by mild deacylation reaction of 37:4 PI(4)P (sn-1 17:0, sn-2 20:4 PI(4)P) (see Figure 1) are shown as representative examples of the LPIP1 synthesis reaction. Figure 3A shows the accurate mass measurement results of 17:0 LPIP ₁ (first row) and its fragment ion (second row), 20:4 LPIP ₁ (third row) and its fragment ion (fourth row) together with the determined molecular formula.

In the reaction products, a substance with m/z = 754.3902 (first stage) and a substance with m/z = 788.3749 (third stage) were detected. The error between these and the theoretical m/z values calculated from the molecular formulas of 17:0 LPIP1 (3-methylated, ethylamine adduct) and 20:4 LPIP1 (3-methylated, ethylamine adduct) was 0.03 ppm (5 ppm or less), and 0.47 ppm (5 ppm or less), respectively, which revealed that substances with the same composition formulas as 17:0 LPIP1 and 20:4 LPIP1 were produced by the above deacylation reaction.

Next, the second row shows the results of detecting substances (fragment ions) produced by decomposition of substances that can be detected in the range of m/z = 754.3902 ± 1.0, including the substances detected in the first row, and the fourth row shows fragment ions of substances that can be detected in the range of m/z = 788.3749 ± 1.0, including the substances detected in the third row.

The substance m/z=754.3902 shown in the first row has m/z 327.2892 and m/z 383.0507 detected in the second row as partial structures. The error between m/z 327.2892 and the theoretical m/z value calculated from the molecular formula of 17:0 monoacylglycerol (17:0 MG) (a monovalent positive ion with one hydroxyl group missing) was 0.48 ppm (5 ppm or less), and the error between m/z 383.0507 and the theoretical m/z value calculated from the molecular formula of inositol diphosphate (detected as a trimethylated, proton adduct) was 1.16 ppm (5 ppm or less). Thus, the structural formula of the substance in the first row was confirmed, and m/z=754.3902 was confirmed to be 17:0 LPIP1 (detected as a trimethylated, ethylamine adduct).

On the other hand, the substance m/z=788.3749 shown in the third row has m/z 361.2736 and m/z 383.0511 detected in the fourth row as its partial structure. The error between m/z 361.2736 and the theoretical m/z value calculated from the molecular formula of 20:4 monoacylglycerol (20:4 MG) (a monovalent positive ion with one hydroxyl group missing) was 0.45 ppm (5 ppm or less), and the error between m/z 383.0511 and the theoretical m/z value calculated from the molecular formula of inositol diphosphate (detected as a trimethylated, proton adduct) was 2.18 ppm (5 ppm or less). Thus, the structural formula of the substance in the third row was confirmed, and m/z=788.3749 was confirmed to be 20:4 LPIP1 (detected as a trimethylated, ethylamine adduct).

The identification of LPIP2 will be described with reference to the chromatogram in Figure 3C and the structural formulae of the reaction starting material, product, and fragment in Figure 3D. Figure 3C shows the results of accurate mass analysis of LPI(4,5)P2 and fragments derived from LPI(4,5)P2 for two types of LPI(4,5)P2 produced by mild deacylation of 37:4 PI(4,5)P2 (sn-1 17:0, sn-2 20:4 PI(4,5)P2) as a representative example of LPIP2 synthesis reaction. The results of accurate mass measurement of 17:0 LPIP ₂ (first row) and its fragment ion (second row), 20:4 LPIP ₂ (third row) and its fragment ion (fourth row) are shown together with the determined molecular formula.

In the reaction products, a substance with m/z = 862.3916 (first stage) and a substance with m/z = 896.3695 (third stage) were detected. The error between these and the theoretical m/z values calculated from the molecular formulas of 17:0 LPIP2 (5-methylated, ethylamine adduct) and 20:4 LPIP2 (detected as 5-methylated, ethylamine adduct) was 4.35 ppm (5 ppm or less) and 3.00 ppm (5 ppm or less), respectively, which revealed that substances with the same composition formulas as 17:0 LPIP2 and 20:4 LPIP2 were produced by the above reaction.

Next, the second row shows the results of detecting substances (fragment ions) produced by decomposition of substances that can be detected in the range of m/z = 862.3916 ± 1.0, including the substances detected in the first row, and the fourth row shows fragment ions of substances that can be detected in the range of m/z = 896.3695 ± 1.0, including the substances detected in the third row.

The substance m/z=862.3916 shown in the first row has m/z=327.2894 and m/z=491.0487 detected in the second row as partial structures. The error between m/z=327.2894 and the theoretical m/z value calculated from the molecular formula of 17:0 monoacylglycerol (17:0 MG) (a monovalent positive ion with one hydroxyl group missing) was 0.01 ppm (less than 5 ppm), and the error between m/z=491.0487 and the theoretical m/z value calculated from the molecular formula of inositol triphosphate (detected as a 5-methylated product and a proton adduct) was 1.69 ppm (less than 5 ppm). Thus, the structural formula of the substance in the first row was confirmed, and it was confirmed that m/z=862.3916 is 17:0 LPIP2 (detected as a 5-methylated product and an ethylamine adduct).

On the other hand, the substance shown in the third row, m/z=896.3695, has partial structures of m/z=361.2737 and m/z=491.0488, which are detected in the fourth row. The error between m/z=361.2737 and the theoretical m/z value calculated from the molecular formula of 20:4 monoacylglycerol (20:4 MG) (a monovalent positive ion with one hydroxyl group missing) is 0.11 ppm (less than 5 ppm), and the error between m/z=491.0488 and the theoretical m/z value calculated from the molecular formula of inositol triphosphate (detected as a 5-methylated, proton-added product) is 1.88 ppm (less than 5 ppm). Therefore, the structural formula of the third substance was confirmed, and m/z=896.3695 was confirmed to be 20:4 LPIP2 (detected as a trimethylated product and an ethylamine adduct).

The identification of LPIP3 will be described with reference to the chromatogram in Figure 3E and the structural formulae of the reaction starting material, product, and fragment in Figure 3F. Figure 3E shows the results of accurate mass analysis of LPI(3,4,5)P3 and fragments derived from LPI(3,4,5)P3 for two types of LPI(3,4,5)P3 generated by mild deacylation reaction of 37:4 PI(3,4,5)P3 (sn-1 17:0, sn-2 20:4 PI(3,4,5)P3). The results of accurate mass measurement of 17:0 LPIP ₃ (first row) and its fragment ion (second row), 20:4 LPIP ₃ (third row) and its fragment ion (fourth row) are shown together with the determined molecular formula. A substance with m/z=970.3843 (first stage) and a substance with m/z=1004.3726 (third stage) were detected in the reaction products. The error between these and the theoretical m/z values calculated from the molecular formulas of 17:0 LPIP3 (7-methylated product, ethylamine adduct) and 20:4 LPIP3 (7-methylated product, ethylamine adduct) was 1.18 ppm (5 ppm or less), and 2.72 ppm (5 ppm or less), respectively, which revealed that substances with the same composition formulas as 17:0 LPIP3 and 20:4 LPIP3 were produced by the above reaction.

Next, the second row shows the results of detecting substances (fragment ions) produced by decomposition of substances that can be detected in the range of m/z = 970.3843 ± 1.0, including the substances detected in the first row, and the fourth row shows fragment ions of substances that can be detected in the range of m/z = 1004.3726 ± 1.0, including the substances detected in the third row.

The substance m/z=970.3843 shown in the first row has m/z=327.2891 and m/z=599.0455 detected in the second row as partial structures. The error between m/z=327.2894 and the theoretical m/z value calculated from the molecular formula of 17:0 monoacylglycerol (17:0 MG) (a monovalent positive ion with one hydroxyl group missing) was 0.85 ppm (less than 5 ppm), and the error between m/z=599.0455 and the theoretical m/z value calculated from the molecular formula of inositol tetraphosphate (detected as a 7-methylated, proton adduct) was 0.01 ppm (less than 5 ppm). Thus, the structural formula of the substance in the first row was confirmed, and it was confirmed that m/z=970.3843 is 17:0 LPIP3 (detected as a 7-methylated, ethylamine adduct).

On the other hand, the substance m/z=1004.3726 shown in the third row has m/z=361.2735 and m/z=599.0444 detected in the fourth row as partial structures. The error between m/z=361.2735 and the theoretical m/z value calculated from the molecular formula of 20:4 monoacylglycerol (20:4 MG) (detected as a monovalent positive ion with one hydroxyl group missing) is 0.62 ppm (less than 5 ppm), and the error between m/z=599.0444 and the theoretical m/z value calculated from the molecular formula of inositol tetraphosphate (detected as a 7-methylated, proton-added product) is 1.94 ppm (less than 5 ppm). Therefore, the structural formula of the third substance was confirmed, and it was confirmed that m/z=1004.3726 was 20:4 LPIP3 (detected as a 7-methylated product, ethylamine adduct).

Figures 3A to 3F above prove the identification of LPIPs by accurate mass measurement.

(Discussion)
By determining the exact masses of the product and the product-derived fragment structures (fragments), the product structures were unambiguously determined as described in the results above. The results obtained by the "measurement and detection method using a hybrid quadrupole-orbitrap mass spectrometer" showed that LPIPs were generated by appropriate methylamine treatment of PIPs, as did the "measurement and detection method by SRM with a triple quadrupole mass spectrometer", and both measurement methods were considered to be suitable for detection and measurement of LPIPs.

(NMR analysis of LPIPs)
The structures of the synthesized LPIPs were determined by NMR. Those skilled in the art can appropriately determine the structure of lipids by NMR, and for example, ¹ H chemical shifts can be found in Ong et al., Mol. Biosys. (2009) 5, 288-298, etc.

In the glycerol skeleton, the 1st position gives a chemical shift of approximately 3.7 ppm, the 2nd position gives a chemical shift of approximately 5.2 ppm, and the 3rd position gives a chemical shift of approximately 4.1 ppm, making it possible to distinguish between sn-1 and sn-2.

At the a- and b-positions of fatty acids, _aCH2 gives a chemical shift of about 2.3 ppm and _bCH3 gives a chemical shift of about 1.6 ppm, making it possible to distinguish between sn-1 and sn-2.

The polyunsaturated portions of polyunsaturated fatty acids give chemical shifts of approximately 2.8 ppm and approximately 5.3 ppm, and can therefore be used to estimate the type of fatty acid in lysophospholipids.

The phosphorus atom gives a chemical shift of 0 to 2 ppm ( ³¹ P, a downfield shift of about 0.5 ppm associated with lysophospholipid formation), and can therefore be used to investigate the progress of a reaction.

For example, the structure of lipids can be determined from NMR as shown in Figure 4A.

NMR measurements were performed on the following samples: NMR measurements were also performed on 1 mg of Brain PI4P (Avanti Polar Lipids, Alabama, USA).
Sample 1) 16:0 LPI4P 5-7 nmol
Sample 2) 17:0 LPI (4,5) P2 4 to 6 nmol
Sample 3) 18:0 LPI(4,5)P2 4-6 nmol
Sample 4) 32:0 (16:0/16:0) PI4P 10 nmol
Sample 5) 37:4 (17:0/20:4) PI(4,5)P2 10nmol
Sample 6) Brain PI (4,5) P2 (mostly 38:4 (18:0/20:4)) 10 nmol
Samples 1-3 were prepared according to the above examples. Samples 4-6 were purchased from Avanti Polar Lipids (Alabama, USA).

About 500 mL of CDCl ₃ :CD ₃ OD=1:1 was added to each of samples 1 to 6, and dissolved using a vortex. ¹ H-1D and TOCSY spectra were measured (37° C., about half a day). 100 mL of EDTA-2Cs solution in D ₂ O at pH=6.0 was added (J. Lipids Res. (1988) 29, 679-689). ³¹ P-NMR spectrum was measured (37° C., about 2 days). Here, only for sample 6, ¹ H-1D and TOCSY spectrum were measured at 25° C. The NMR measurement time for 1 mg of Brain PI4P was about 1/10 of the other samples, and about 1/100 of the ³¹ P-1D.

NMR analysis was performed on Brain PI4P to assign the inositol carbon positions, and the inositol carbon positions were successfully assigned.

In sample 4 (32:0 PI4P), signals from fatty acids and C1, C2, and C3 of the glycerol backbone were observed, but no signals from polyunsaturated fatty acids were observed.

In sample 6 (Brain PI(4,5)P2), signals from fatty acids and C1, C2, and C3 of the glycerol backbone were observed, but almost no signals from polyunsaturated fatty acids were observed.

As a result of the measurement, it was confirmed that most of the signals were derived from the target compound. The structures of Samples 1 to 3 and 5 confirmed by NMR are shown in Figures 4B to 4F.
(Conclusion)
In NMR, with a sample amount of about 5 to 10 nmol, it was possible to observe signals of at least a part of the fatty acid and glycerol backbone of the lysophospholipid, and it was possible to determine the structure of the lysophospholipid. Also, with a sample amount of about 50 nmol, it was possible to observe signals of all hydrogen atoms and phosphorus atoms.

(Example 2: Analysis of biological samples and isolation of LPIPs (cultured cells))
In this example, an experiment was carried out on the separation and analysis of LPIPs in a biological sample, using a human cultured cell line as the biological sample.

(material and method)
(Cultured cell lines) HEK293T (human fetal kidney epithelial cell line, Open biosystems catalog number HCL4517) and Jurkat (human acute T-cell leukemia cell-derived cell line, ATCC catalog number TIB-152) were cultured and maintained under conditions recommended by the supplier.

(Extraction of phospholipids) Lipid extraction was performed based on the Bligh & Dyer method (EG Bligh et al, Canadian Journal of Biochemistry and Physiology, 1959, 37(8): 911-917, 10.1139/o59-099).

For the analysis of PIPs, cultured cells and mouse tissues were scraped or homogenized in 1.5 mL of ice-cold methanol, and 2 μL of 1 nmol/μL 8:0/8:0-PI(4,5)P ₂ was added as an anti-adhesive agent, followed by 200 fmol/μL 17:0/20:4-PI4P, 17:0/20:4-PI(4,5)P _2, 17:0/20:4-PI(3,4,5)P ₃ , 17:0/20:4-PI, 17:1-LPI, and 17:0/20:4-PS. 50 μL was added as an internal standard, 2N HCl (0.75 mL), water (0.75 mL), 1M NaCl (0.2 mL) and chloroform (3 mL) were added, followed by stirring and centrifugation. After centrifugation, the lower layer was collected.

(Separation with DEAE cellulose)
Before loading the phospholipids extracted by the above procedure onto the DEAE-cellulose column, 1.5 mL of methanol was added to each sample. The lipids adsorbed on the column were washed sequentially with chloroform/methanol (1:1, v/v) (3 mL) and chloroform/methanol/28% aqueous ammonia/acetic acid (200:100:3:0.9, v/v) (3 mL). The highly acidic lipids including LPIPs were then eluted with chloroform/methanol/HCl/water (12:12:1:1, v/v) (1.5 mL). After the addition of water (0.75 mL) and 1 M NaCl (0.1 mL), the solution was stirred, centrifuged, and the lower layer was collected.

(Methylation of phosphate groups)
After the addition of 150 μL of 0.6 M TMS-diazomethane in hexane, the samples were incubated at room temperature for 10 min (Nature Methods 8, 267-272 (2011)). Glacial acetic acid (15 μL) and washing solution (0.7 mL of chloroform/methanol/water (3:48:47, v/v)) were then added to each solution. The solutions were vortexed, centrifuged, and the bottom layer was collected. Each sample was dried under nitrogen gas, redissolved in 24 μL of methanol/70% ethylamine (100:0.065, v/v), and 8 μL of water was added. The resulting acidic phospholipid-rich fractions, including LPIPs, were analyzed within 1 day of preparation.

(Analysis Method)
The measurement was performed by the "measurement and detection method by SRM with a triple quadrupole mass spectrometer" with excellent quantitative performance. Based on the molecular species analysis of diacyl type PIPs, selective reaction monitoring (SRM) was performed for LPIPs molecular species expected to be contained in biological samples. Using a TSQ Vantage, Q1: m/z of parent ion and Q3: m/z of monoacylglycerol, which is a product ion. Mass spectrometry data was quantified by peak area, and the abundance of each molecular species was calculated based on LPI added as an internal standard substance. The adduct ion was [M+CH ₃ CH ₂ NH ₃ ] ⁺ . Table 5 shows a list of m/z values designated by Q1 and Q3 used for the detection of each LPIPs in this example.

(Analysis Results)
Figure 5A shows the presence of LPIP1, LPIP2, and LPIP3 in HEK293T cells, and Figure 5B shows the presence of LPIP3 in Jurkat cells. The horizontal axis shows molecular species with different fatty acid compositions, and the vertical axis shows the amount present in cell-derived phospholipids containing 1 nmol of PS. LPIPs with various acyl groups with 16 to 22 carbon atoms and 0 to 6 double bonds were found in the cells. LPIP1 and LPIP2 were trace phospholipids present at a level of about one hundredth to one tenth of that of PS, and LPIP3 was present at a level of about one hundredth to one thousandth of that of PS.

In addition, the results of analysis of lipid extracts from HEK293T cells using a "measurement and detection method using a hybrid quadrupole-orbitrap mass spectrometer" to detect 18:0 LPIP3 and its specific fragments are shown in Figures 5C and 5D.

As shown in the upper part of Figure 5C, a peak at m/z = 984.4024 was detected, and the error from the theoretical m/z value (984.4012) of 18:0 LPIP3 (7-methylated, methylamine added) was 1.2673 ppm (less than 5 ppm), indicating that a substance with the same composition as 18:0 LPIP3 was contained in the lipid extract of HEK293 cells. As shown in the lower part of Figure 5D, peaks at m/z 341.3047 and m/z 599.0449 were detected as fragments of ions detected in the range of m/z = 984.4024 ± 0.2. The former corresponds to the m/z of 18:0 monoacylglycerol (a monovalent positive ion with one hydroxyl group missing), and the latter corresponds to the m/z of inositol tetraphosphate (7-methylated, protonated), and both have an error of 5 ppm or less from the theoretical value (1.0460 and 1.0539) (see FIG. 3E). The latter peak is the same ion as the fragment ion generated from the LPIP3 synthesis product. If m/z 341.3047 and m/z 599.0449 in the lower part of FIG. 5C are ion fragments derived from m/z = 984.4024 in the upper part of FIG. 5D, the times at which these peaks are detected are the same. FIG. 5D shows that m/z 341.3047 and m/z 599.0449 are detected at the same elution time as m/z = 984.4024.

_{The CH3CH2NH3 +} ^adduct _is shown below.

Before the addition it looks like this:

The dehydroxylated group is shown below.

Shown below before dehydroxylation.

The H ⁺ adduct is shown below.

Before the addition it looks like this:

Example 3: Analysis and isolation of LPIPs in biological samples (mouse tissue and human blood samples)
Next, in this Example, an experiment on the separation and analysis of LPIPs in biological samples was carried out using other samples, such as mouse or human tissue, serum, or plasma.

Mouse: C57BL/6J (purchased from Japan CLEA Co., Ltd.), 12-16 weeks old Organs/tissues: Brain, heart, liver, large intestine were prepared as follows. Mice were cervically dislocated, fixed in a supine position, and then abdominally opened. Organs isolated after perfusion with PBS and blood removal were promptly frozen in liquid nitrogen. They were thawed just before lipid extraction and homogenized with a homogenizer. Phospholipid extraction, separation with DEAE cellulose, and methylation reaction were performed according to Example 2. Analysis was also performed using the "Measurement and detection method by SRM with a triple quadrupole mass spectrometer" as in Example 2. Serum: Three minutes after intraperitoneal administration of an anesthetic (ketamine), the abdomen was opened, and blood was collected from the heart. The collected blood was left at room temperature for 15 minutes, and then at 4°C for 12 hours. Blood clots were removed by centrifugation (600g, 30 minutes), and serum was prepared. The extraction of phospholipids, separation with DEAE cellulose, and methylation reaction were carried out in accordance with Example 2. Analysis was also carried out in the same manner as in Example 2, using the "measurement and detection method by SRM with a triple quadrupole mass spectrometer".

Plasma: Three minutes after intraperitoneal administration of an anesthetic (ketamine), the abdomen was opened and 0.5 ml of blood was collected from the lower abdominal aorta. The collected blood was collected in an ice-cold 1.5 mL tube containing 2 mg EDTA-2Na, and the supernatant after stirring and centrifugation (600 g, 30 minutes) was used to prepare a plasma sample. Phospholipid extraction, separation with DEAE cellulose, and methylation reaction were performed according to Example 2. Analysis was also performed using the "Measurement and detection method by SRM with a triple quadrupole mass spectrometer" as in Example 2.

Human serum: Blood was collected from a peripheral vein into a test tube and left to stand at room temperature for 15 minutes, then at 4°C for 12 hours. Blood clots were removed by centrifugation (600g, 30 minutes) to prepare serum. Phospholipid extraction, separation with DEAE cellulose, and methylation reaction were performed as in Example 3. Analysis was performed using the "Measurement and detection method by SRM with a triple quadrupole mass spectrometer" as in Example 2.

(result)
As shown in Figure 6A, it was revealed that LPIPs exist in mouse tissues. Furthermore, since LPIPs exist as a liquid component in the body, such as serum shown in Figure 6B and plasma shown in Figure 6C, it was suggested that LPIPs produced in cells are secreted, or that production and decomposition of LPIPs (for example, production of LPIP2 by phosphorylation of LPIP1) also occurs outside the cells. As shown in Figure 6D, LPIPs was also present in human serum.

(Discussion)
In addition to samples from mice and humans, other experiments have revealed that LPIPs are also present in yeast and Drosophila, demonstrating that LPIPs are present both intracellularly and extracellularly in many biological species.

(Example 4: Use as a cancer marker)
In this example, the usefulness of novel LPIPs was examined.

(material and method)
PTEN ^flox/flox mice (control mice) and PB-Cre4:PTEN ^flox/flox mice (prostate-specific Pten-deficient mice), male, 16 weeks old
Organ: Prostate Samples were prepared as follows. Mice were cervically dislocated and fixed in a supine position, and then the abdomen was opened. The prostate isolated under a stereomicroscope was quickly frozen in liquid nitrogen. Just before lipid extraction, the prostate was thawed and homogenized with a homogenizer. Phospholipid extraction, separation with DEAE cellulose, and methylation reaction were performed according to Example 2. Analysis was also performed by the "measurement and detection method by SRM with a triple quadrupole mass spectrometer" as in Example 2.

(result)
In FIG. 7A, the upper part shows tissue weight and the lower part shows tissue image by HE staining for the prostate of a normal mouse (Ctrl) or a mouse (PTEN KO) that specifically lacks the tumor suppressor gene Pten in the prostate. It can be seen that Pten deficiency develops prostate cancer. As shown in FIG. 7B, it was revealed that 16:0 LPIPs was elevated in prostate cancer tissue compared to the control normal prostate. In particular, LPIP3 was found to be significantly elevated with tumor formation, while it was below the detection limit in normal tissue. Since the fluctuation of LPIPs correlates with carcinogenesis, the usefulness of LPIPs as a cancer treatment target or a biomarker reflecting cancer is expected.

(Example 5: Use as an inflammation marker)
(material and method)
C57BL/6J mice (purchased from CLEA Japan, Inc.), aged 12-16 weeks, were dissected after cervical dislocation to obtain femurs. Bone marrow cells and neutrophils were collected from the obtained femurs and stimulated with complement component C5a, an inflammatory substance. Phospholipid extraction, separation with DEAE cellulose, and methylation reaction were performed in the same manner as in Example 2. Analysis was also performed in the same manner as in Example 2 using the "measurement and detection method by SRM with a triple quadrupole mass spectrometer".

(result)
As shown in Figure 8, 18:0 LPIP3 increases with stimulation by complement component C5a, which is involved in the activation of blood cells related to inflammation such as neutrophils and macrophages. It has been suggested that LPIP3 may be involved in the chemotaxis of inflammatory cells, production of active oxygen, phagocytosis, enzyme secretion reactions, etc., and it is expected to be useful as a therapeutic target for inflammation.

(Example 6) Separation of Phosphoinositides by Column In this example, it was demonstrated that separation of phosphoinositides (PIPs) based on the position of the phosphate group by LC-MS is possible.

(material and method)
(Materials used)
Phosphatidylinositol phosphates (phosphoinositides) used as samples for the following analyses were purchased from Avanti Polar Lipids (Alabama, USA).
17:0-20:4 PI(3)P(LM-1900)
17:0-20:4 PI(4)P(LM-1901)
17:0-20:4 PI(5)P(LM-1902)
17:0-20:4 PI (3,4) P2 (LM-1903)
17:0-20:4 PI (4,5) P2 (LM-1904)
17:0-20:4 PI (3,5) P2 (LM-1905)
17:0-20:4 PI(3,4,5)P3(LM-1906)
17:0-20:4 PI (LM-1502)
Other glycerophospholipid standard synthesis All solvents utilized were of HPLC or LC-MS grade, and other chemical reagents were of analytical grade. Trimethylsilyldiazomethane (TMS-diazomethane) was purchased from Tokyo Kasei. Ultrapure water was obtained from Kanto Chemicals (Tokyo, Japan).

Each phosphoinositide was dissolved in chloroform:methanol = 1:1 to a concentration of 100 μM in a glass vial (4 mL size) and stored in a freezer at -30°C.

The following samples were prepared from this storage solution.

Each phosphoinositide alone (100 μM 1 μL)
PI(3)P + PI(4)P + PI(5)P (1 μL each, 100 μM)
PI(3,4)P2+PI(4,5)P2+PI(3,5)P2 (100 μM 1 μL each)
Each sample was treated with TMS diazomethane to protect the phosphate group. Specifically, each sample was treated with 150 μL of 0.6 M TMS diazomethane (22-25° C.), and 5 minutes later, 15 μL of glacial acetic acid was added to stop the methylation reaction. Then, 700 μL of a mixture of chloroform:methanol:water (3:48:47) was added to each sample, and after centrifugation, the organic layer (lower layer) was dispensed into a Spitz test tube, evaporated to dryness using a nitrogen gas concentrator, and redissolved in 100 μL of acetonitrile to prepare a sample for LC-MS measurement.

(Measurement conditions)
The LC-MS measurement was carried out under the following conditions.

(Equipment used)
Mass spectrometer: QTRAP6500 with SelexION System (ABSciex, Tokyo, Japan)
Pump: Nexera X2 system (Shimadzu Corporation, Kyoto, Japan)
Autosampler: PAL HTC-xt (AMR, Tokyo, Japan)
Column: CHIRALPAK IC-3, 2.1 mm x 250 mm, particle size 3 μm (DAICEL corporation, Osaka, Japan)
Flow rate: 0.1 mL/min. Sample volume injected: 10 μL.
(Chromatography conditions)
Mobile phase A: acetonitrile + 5 mM ammonium acetate Mobile phase B: methanol + 5 mM ammonium acetate *All of these reagents were purchased from Wako Pure Chemical Industries (Tokyo, Japan) and were of LC-MS grade.

Gradient conditions (linear gradient)
0 min, mobile phase B = 40%
1 minute, mobile phase B=40%
3 minutes, mobile phase B=85%
14 minutes, mobile phase B=85%
15 minutes, mobile phase B=40%
20 minutes, mobile phase B=40%
Mass spectrometer conditions Ionization method: Electrospray (positive ion mode)
Measurement method: Multiple reaction monitoring (MRM)
Control software: Analyst 1.6.2 (Sciex, Tokyo, Japan)
The following measurement methods were used:

Software: Software Version: Analyst 1.6.2 Figure 1 shows the chromatograms obtained by measuring C17:0/C20:4-PI(3)P, -PI(4)P, and -PI(5)P as internal surrogate standards, and the chromatograms obtained by measuring C17:0/C20:4-PI(3,5)P2, -PI(3,4)P2, and -PI(4,5)P2. The three isomers of PIP1 were eluted and separated in the order of PI(3)P, PI(4)P, and PI(5)P. The three isomers of PIP2 were eluted and separated in the order of PI(3,5)P2, PI(3,4)P2, and PI(4,5)P2.

The combinations of Q1 precursor ions and Q3 product ions used are shown below.

<Mass spectrometry measurement conditions>
Parameter Table (Period1 Experiment1)
CUR: 20.00
CAD: 9.00
IS:5500.00
TEM:100.00
GS1: 50.00
GS2: 50.00
D.P. 100.00
EP 10.00
CXP 12.00
Resolution tables
Quad1 Positive Unit Scan Speed=10Da/s
(Calibration conditions)
Polypropylene glycol (PPG) was used as a calibration reagent, with the following results:
IE1 1.800
Mass(Da) Offset Value
59.050 0.010
175.133 -0.035
500.380 -0.194
616.464 -0.268
906.673 -0.421
1254.925 -0.617
1545.134 -0.782
1952.427 -1.013
Quad3 Positive Unit Scan Speed=10Da/s
IE3 1.000
Mass(Da) Offset Value
59.050 0.005
175.133 -0.012
500.380 -0.085
616.464 -0.105
906.673 -0.183
1254.925 -0.270
1545.134 -0.329
1952.427 -0.429
Calibration tables
Quad1 Positive Unit Resolution Scan Speed=10Da/s
Mass(Da) Dac Value
175.133 18682
500.380 53678
616.464 66162
906.673 97393
1254.925 134868
1545.134 166099
1952.427 209933
Quad3 Positive Unit Resolution Scan Speed=10Da/s
Mass(Da) Dac Value
59.050 6189
175.133 18637
500.380 53564
616.464 66028
906.673 97195
1254.925 134603
1545.134 165759
1952.427 209490
(Apparatus parameters)
Detector Parameters (Positive):
CEM 1800.0
(Software version)
Software version: Analyst 1.6.2
The data thus obtained was analyzed using Analyst (Sciex, Tokyo, Japan) or Multi Quant (Sciex, Tokyo, Japan).

The results of isomer separation using the 17:0/20:4 synthetic product are shown in Figure 1. Phosphoinositides with different numbers of phosphate groups were eluted at different retention times. Even when three isomers differing only in the position of the phosphate group were mixed (a mixture of PIP or PIP2), they were successfully separated using the column in a 20-minute measurement method. In this way, separation of phosphoinositides by phosphate group position isomers enables position-specific analysis.

It was also confirmed that it is possible to identify the combination of types of acyl groups in phosphoinositides by using the results of MS and MS/MS measurements (see Clark J et al., Nat Methods. 2011 Mar;8(3):267-72. doi:10.1038/nmeth for the identification of types of acyl groups). Therefore, the method of the present invention can be used to identify the position of the phosphate group and the type of acyl group in phosphoinositides.

Example 7 Quantification of Phosphoinositides To investigate the accuracy of quantification of phosphoinositides by LC-MS/MS, the effects of different sample amounts and repeated measurements were examined.

Sample Preparation Synthetic 17:0/20:4 phosphoinositide samples were prepared as in Example 6.

The samples were subjected to the following methylation treatment. The samples were treated with 150 μL of 0.6 M TMS diazomethane (22-25°C), and after 5 minutes, 15 μL of glacial acetic acid was added to stop the methylation reaction. Then, 700 μL of a mixture of chloroform:methanol:water (3:48:47) was added to each sample, and after centrifugation, the organic layer (lower layer) was dispensed into a Spitz test tube and evaporated to dryness using a nitrogen gas concentrator. Each was then redissolved in 100 μL of acetonitrile to prepare samples for LC-MS measurement.

Eight types of 17:0/20:4 phosphoinositides (PI(3)P, PI(4)P, PI(5)P, PI(3,4)P2, PI(4,5)P2, PI(3,5)P2, PI(3,4,5)P3 and PI) were injected at 0.02, 0.05, 0.1, 0.2, 5, 10 and 50 pmol (each injection amount was repeated four times) and the signal intensities (peak areas) were compared when measured by LC-MS. The calibration curve results are shown in Figure 2.

The calculation formulas for each calibration curve are shown in the table below.

The calibration curve for C17:0/C20:4-PIPs was linear in the range of 0.002 pmol to 50 pmol. The limits of detection (LOD) were 0.005-0.033 pmol, and the limits of quantification (LOQ) were 0.014-0.099 pmol.

These results confirm that good quantitative results can be obtained even for trace amounts of phosphoinositides at or below the pmol level.

Similarly, to investigate the variability between measurement runs for each phosphoinositide, 50 replicate measurements (injection amount of sample: 10 pmol) were performed for each sample, and the results are shown in Figure 3. The coefficients of variance were 8.4-12.0%, indicating that the variability between measurement runs was small.

Phosphoinositides are stable in the prepared samples, and it is predicted that the effect of differences in measurement timing between samples on quantitative results is small. In other words, these results indicate that the developed analytical method has excellent quantitative properties and reproducibility.

Example 8 Separation of Phosphoinositides by Ion Mobility Separation Example 8 demonstrates that separation of phosphoinositides based on the position of their phosphate group is possible by ion mobility separation.

PIP2 isomers were separated using a method other than chiral column (mass spectrometry only). Using ion mobility, we attempted to separate intact PIP2 (C17:0/C20:4-PI(3,5)P2, -PI(3,4)P2 and -PI(4,5)P2). Details are given below.

The following phosphoinositides were purchased from Avanti Polar Lipids (Alabama, USA):
17:0-20:4 PI (3,4) P2 (LM-1903)
17:0-20:4 PI (4,5) P2 (LM-1904)
17:0-20:4 PI (3,5) P2 (LM-1905)
From this, a sample for MS analysis was prepared in the same manner as in Example 6.

The measurements were carried out under the following conditions:
MS:
Apparatus: Q TRAP 6500, SelexION system (ABSciex, Tokyo, Japan)
Control software: Analyst 1.6.2, PeakView (Sciex, Tokyo, Japan)
Ionization/ion source: ESI/IonDrive (Sciex, Tokyo, Japan)
Syringe pump: 7 μL/min Ion source parameters:
Curtain gas: 20
Collision Gas: Medium
Ion spray voltage: 5500 V (positive ion mode)
Temperature: 500°C
Ion source gas 1: 70 psi
Ion source gas 2: 50 psi
The results are shown in Figure 12. As shown in Figure 12, PI(4,5)P2 could be separated from PI(3,5)P2 and PI(3,4)P2 by changing the compensation voltage (COV). This demonstrated that positional isomers of phosphoinositides can be separated without using a column. The C17:0/C20:4-PIP2 used in the measurement was not methylated.

Further experiments combining ion mobility with LC demonstrated more selective detection of phosphoinositides.

Example 9: Analysis to assess changes in phosphoinositide composition
This example shows that the method of the present invention can be used to evaluate changes in phosphoinositide composition in cells treated with a drug. As in Example 6, the elution order of positional isomers of phosphate groups was confirmed in advance for each diacylglycerol species.

HEK293T cells (human embryonic kidney epithelial cell line, Open biosystems (Colorado, USA), catalog number HCL4517 ₎ were cultured under the conditions recommended by the supplier until ^1x106 cells/10 cm dish (day 0). On day 2, _H2O2 was added to the culture medium to a final concentration of 10 mM, and the cells were scraped and collected with a cell scraper at 0, 2, 5, and 15 minutes, respectively. The pellets were collected after centrifugation at 800g for 3 minutes, and further washed with PBS(-) to collect the cells.

Then, the following lipid extraction and methylation treatment were carried out and measured by chiral LC-MS/MS.
Phospholipid Extraction For PIPs analysis, cell samples were spiked with 50 μL of 0.2 pmol/μL 17:0/20:4-PI3P, 17:0/20:4-PI4P, 17:0/20:4-PI5P, 17:0/20:4-PI(3,5)P2, 17:0/20:4-PI(3,4)P2, 17:0/20:4-PI(4,5)P2, 17:0/20:4-PI(3,4,5)P3, 2 pmol/μL 17:0/20:4-PI, and 17:0/20:4-PS, which served as internal surrogate standards. Next, 2 μL of 1 nmol/μL 8:0/8:0-PI(4,5)P2 (as an anti-adsorption agent), 0.75 mL of 2N HCl, 0.75 mL of water, 0.2 mL of 1M NaCl, and 3 mL of chloroform were added and shaken. After centrifugation, the lower layer was collected, and 1.5 mL of methanol was added to each sample, followed by application to a DEAE-cellulose column (Santa Cruz Biotechnology, Texas, USA). The lipids adsorbed on the column were washed sequentially with chloroform:methanol (1:1, v/v) (3 mL) and chloroform:methanol:28% aqueous ammonia:acetic acid (200:100:3:0.9, v/v) (3 mL). Highly acidic lipids, including PIPs, PI, and PS, were then eluted with chloroform:methanol:HCl:water (12:12:1:1, v/v) (1.5 mL). After adding 0.75 mL of water and 0.1 mL of 1 M NaCl, the solution was shaken, centrifuged, and the bottom layer was collected. After adding 150 μL of 0.6 M TMS-diazomethane in hexane, the samples were incubated for 10 min at room temperature (Nature Methods). Then, 20 μL of glacial acetic acid and 0.7 mL of a washing solution (chloroform:methanol:water (3:48:47, v/v)) were added to each solution. The solution was shaken, centrifuged, and the bottom layer was collected. Each sample was dried under nitrogen gas and redissolved in acetonitrile. The resulting PIPs-enriched fractions were analyzed within one day of preparation.

The methylation was carried out by treating each sample with TMS diazomethane as in Example 6.
LC-ESI MS/MS System Sample analysis was carried out under the following conditions as in Example 6.
Mass spectrometer: QTRAP 6500 (ABSciex, Tokyo, Japan)
Pump: Nexera X2 system (Shimadzu Corporation, Kyoto, Japan)
Autosampler: PAL HTC (CTC Analytics, Zwingen, Switzerland)
Column: CHIRALPAK IC-3, 2.1 mm x 250 mm, particle size 3 μm (DAICEL corporation, Osaka, Japan)
Injector: Non-metallic injector unit Sample loop: 25 μL PEEK tube Flow rate: 0.1 mL/min Sample volume injected: 10 μL
Temperature: Room temperature (chromatographic conditions)
Mobile phase A: acetonitrile + 5 mM ammonium acetate Mobile phase B: methanol + 5 mM ammonium acetate *All of these reagents were purchased from Wako Pure Chemical Industries (Tokyo, Japan) and were of LC-MS grade.

Gradient conditions (linear gradient)
0 min, mobile phase B = 40%
1 minute, mobile phase B=40%
3 minutes, mobile phase B=85%
14 minutes, mobile phase B=85%
15 minutes, mobile phase B=40%
20 minutes, mobile phase B=40%
Mass spectrometer conditions Ionization method: Electrospray (positive ion mode)
Measurement method: Multiple reaction monitoring (MRM)
The measurement results are shown in FIG. 13 and FIG.

The results in Figure 13 show the extracted ion chromatograms of 38:4 phosphoinositides, which indicate that as _{the H2O2} treatment time increases, PI(4,5)P2 decreases and PI(3,4)P2 increases.

The results in Figures 14A to _14D show how PIPs having different diacylglycerols change with _H2O2 treatment. Statistical analysis was performed using one-way analysis _of variance and Tukey's multiple comparison test. It was also shown that for PIPs having different diacylglycerols, PI(4,5)P2 decreased and PI(3,4)P2 increased with increasing _H2O2 treatment time.

Thus, by using the method of the present invention, it is possible to closely observe changes in phosphoinositide composition (type of diacylglycerol, number and position of phosphate groups in PIPs) in drug-treated cells.

Example 10: Evaluation of changes in phosphoinositide composition in genetically engineered mice
This example shows that the method of the present invention can be used to evaluate changes in phosphoinositide composition in genetically engineered mice. As in Example 6, the elution order of positional isomers of the phosphate group for each diacylglycerol species was confirmed in advance.

We observed changes in phosphoinositide composition when two genes involved in phosphoinositide metabolism (INPP4B and PTEN) were manipulated.

INPP4B undergoes the following dephosphorylation reaction: PI(3,4,5)P3 → PI(3,4)P2 → PI(3)P
PTEN promotes the following dephosphorylation reaction PI(3,4,5)P3 → PI(4,5)P2
It has been reported that it promotes the regulating activity of IFN-γ-α, γ-α-carboxylates (Kofuji S et.al., Cancer Discov. 2015 Jul;5(7):730-9, Li Chew C et.al., Cancer Discov. 2015 Jul;5(7):740-51 and Vo TT et.al., Cancer Discov. 2015 Jul;5(7):697-700).

Mice generated according to a previous paper (Kofuji S et.al., Cancer Discov. 2015 Jul;5(7):730-9) were used in the experiment. Specifically, the mice were generated as follows. A conditional targeting vector was constructed to delete a gene fragment containing the 21st coding exon of the mouse Inpp4b gene by homologous recombination. One loxP site was introduced into intron 20 and two loxP sites were introduced into intron 21. A LacZ-PGK-Neo ^r cassette was inserted between the two lox sites in intron 21 in the antisense direction to the transcription of Inpp4b. The linear construct was introduced into ^1x107 E14K mouse embryonic stem (ES) cells by electroporation (Sasaki T et.al., Science 2000;287:1040-6). ES cell colonies resistant to G418 (0.3mg/mL; Life Technologies) were screened by standard PCR to select colonies that had undergone homologous recombination. Recombinant clones were confirmed by standard Southern blotting of HindIII-digested genomic DNA fragments using a 590bp probe. Targeted ES cells were injected into C57BL/6J blastocysts (CLEA Japan, Tokyo, Japan). Chimeric male mice were mated with C57BL/6JJ female mice to achieve germline inheritance. Inpp4b ^{+/ flox mice were generated by crossing with MeuCre40 transgenic mice (Leneuve P et. al., Nucleic Acids Res 2003;31:e21) to generate Inpp4b +/Δ mice, in which the selection cassette had been deleted. Similarly, Inpp4b +/flox} mice were crossed with MeuCre40 mice to generate Inpp4b ^+/Δ mice. PCR confirmed that Inpp4b ^Δ/Δ mice were distinct from Inpp4b ^+/ Δ and Inpp4b ^+/+ mice. An oligo primer common to the Inpp4A ⁺ and Inpp4A ^Δ alleles (5'-CCTGCCATGGGTAGATTTCT-3'), a primer specific to the Inpp4A ⁺ allele (5'-GTTTACATTTGACAGGGTGGTTGG-3'), and a primer specific to the Inpp4A ^Δ allele (5'-TGCTGTCGCCGAAGAAGTTA-3') were combined in the same PCR reaction. ^{Inpp4b +/Δ} Pten ^+/- mice were generated by crossing Inpp4b ^+/Δ mice with Pten ^+/- mice (Suzuki A et al., Curr Biol 1998;8:1169-78). To generate Inpp4b ^Δ/Δ Pten ^+/− mice, Inpp4b ^+/Δ Pten ^+/− mice were crossed with Inpp4b ^+/Δ mice. Triple mutants were generated by crossing Inpp4b ^+/Δ Pten ^+/− mice with Akt1 ^−/− or Akt2 ^−/− mice (purchased from Jackson Laboratory). All animal experiments were performed with the review and approval of the Akita University Animal Care and Use Committee.

Among the mice thus produced, the thyroid glands obtained from the following mice were used as samples.
C57/B6J mice (male or female, 5-6 months old) (N=3)
INPP4B(-/-) mice (male or female, 5-8 months old) (N=4)
PTEN (+/-) mice (male or female, 5-7 months old) (N=4)
INPP4B(-/-)PTEN(+/-) mice (male or female, 5-7 months old) (N=4)
Each sample was subjected to the following lipid extraction and methylation treatment, and then measured by chiral LC-MS/MS.
Phospholipid Extraction For PIPs analysis, each mouse tissue was homogenized with 1.5 mL of ice-cold methanol and 50 μL of 0.2 pmol/μL 17:0/20:4-PI3P, 17:0/20:4-PI4P, 17:0/20:4-PI5P, 17:0/20:4-PI(3,5)P2, 17:0/20:4-PI(3,4)P2, 17:0/20:4-PI(4,5)P2, 17:0/20:4-PI(3,4,5)P3, 2 pmol/μL 17:0/20:4-PI, and 17:0/20:4-PS were added to serve as internal surrogate standards. Next, 2 μL of 1 nmol/μL 8:0/8:0-PI(4,5)P2 (as an anti-adsorption agent), 0.75 mL of 2N HCl, 0.75 mL of water, 0.2 mL of 1M NaCl, and 3 mL of chloroform were added and shaken. After centrifugation, the lower layer was collected, and 1.5 mL of methanol was added to each sample, followed by application to a DEAE-cellulose column (Santa Cruz Biotechnology, Texas, USA). The lipids adsorbed on the column were washed sequentially with chloroform:methanol (1:1, v/v) (3 mL) and chloroform:methanol:28% aqueous ammonia:acetic acid (200:100:3:0.9, v/v) (3 mL). Highly acidic lipids, including PIPs, PI, and PS, were then eluted with chloroform:methanol:HCl:water (12:12:1:1, v/v) (1.5 mL). After adding 0.75 mL of water and 0.1 mL of 1 M NaCl, the solution was shaken, centrifuged, and the bottom layer was collected. After adding 150 μL of 0.6 M TMS-diazomethane in hexane, the samples were incubated for 10 min at room temperature (Nature Methods). Then, 20 μL of glacial acetic acid and 0.7 mL of a washing solution (chloroform:methanol:water (3:48:47, v/v)) were added to each solution. The solution was shaken, centrifuged, and the bottom layer was collected. Each sample was dried under nitrogen gas and redissolved in acetonitrile. The resulting PIPs-enriched fractions were analyzed within one day of preparation.

The methylation was carried out by treating each sample with TMS diazomethane as in Example 6.
LC-ESI MS/MS System Sample analysis was carried out under the following conditions as in Example 6.
Mass spectrometer: QTRAP 6500 (ABSciex, Tokyo, Japan)
Pump: Nexera X2 system (Shimadzu Corporation, Kyoto, Japan)
Autosampler: PAL HTC (CTC Analytics, Zwingen, Switzerland)
Column: CHIRALPAK IC-3, 2.1 mm x 250 mm, particle size 3 μm (DAICEL corporation, Osaka, Japan)
Injector: Non-metallic injector unit Sample loop: 25 mL PEEK tube Flow rate: 0.1 mL/min Sample volume injected: 10 μL
Temperature: Room temperature (chromatographic conditions)
Mobile phase A: acetonitrile + 5 mM ammonium acetate Mobile phase B: methanol + 5 mM ammonium acetate *All of these reagents were purchased from Wako Pure Chemical Industries (Tokyo, Japan) and were of LC-MS grade.

Gradient conditions (linear gradient)
0 min, mobile phase B = 40%
1 minute, mobile phase B=40%
3 minutes, mobile phase B=85%
14 minutes, mobile phase B=85%
15 minutes, mobile phase B=40%
20 minutes, mobile phase B=40%
Mass spectrometer conditions Ionization method: Electrospray (positive ion mode)
Measurement method: Multiple reaction monitoring (MRM)
The measurement results are shown in FIG. 15 and FIG.

Figure 15 shows the extracted ion chromatogram of 38:4 phosphoinositide. PI(3,4)P2 was not observed in wild-type or INPP4B(-/-) mice, but was produced in PTEN(+/-) mice, and was further enhanced in INPP4B(-/-)PTEN(+/-) mice. This result is consistent with the reported activity of PTEN.

The results in Figures 16A-D show how PIPs with different diacylglycerols change with genetic manipulation. Statistical analysis was performed using one-way analysis of variance and Tukey's multiple comparison test. As expected, the amount of PIP3 was greatly increased in mice in which the gene promoting the degradation of PIP3 was knocked out, but no decrease in PIP2 or PIP was observed. PI(3,4)P2 was not observed in wild-type or INPP4B(-/-) mice, but its production was observed in PTEN(+/-) mice, and further enhanced production was observed in INPP4B(-/-)PTEN(+/-) mice.

Thus, by using the method of the present invention, it is possible to closely observe changes in phosphoinositide composition (type of diacylglycerol, number and position of phosphate groups in PIPs) in genetically engineered mice.

(Example 11: Separation and quantification of phosphate positional isomers of LPIPs)
This example demonstrates that phosphate positional isomers of LPIPs can be separated and quantified.

Using the 17:0/20:4 PIPs synthesis product as the starting material, 17:0 lysoPI(3)P, lysoPI(4)P, lysoPI(5)P, lysoPI(3,5)P2, lysoPI(3,4)P2, and lysoPI(4,5)P2 were prepared according to the method of Example 1.

The measurement conditions were the same as in Example 6, except that the combination of the precursor ion of Q1 and the product ion of Q3 was set as follows:
17:0 Rizzo PIP1
Q1: 726.5 (Da) Q3: 327.3 (Da)
17:0 Rizzo PIP2
Q1: 834.5 (Da) Q3: 327.3 (Da)
The results of the analysis performed in the same manner as in Example 6 are shown in Figure 17. Three types of isomers differing only in the position of the phosphate group were observed at different retention times. This shows that separation of lysophosphoinositides based on their phosphate group positional isomers enables quantitative positional isomer-specific analysis. Therefore, the method of the present invention can be used to analyze the position of the phosphate group and the type of acyl group for lysophosphoinositides as well.

(Note)
As described above, the present invention has been illustrated using preferred embodiments of the present invention, but it is understood that the scope of the present invention should be interpreted only by the claims. It is understood that the patents, patent applications and literature cited in this specification should be incorporated by reference to this specification in the same manner as if the contents themselves were specifically described in this specification. This application claims priority to Japanese Patent Application No. 2016-144177 filed on July 22, 2016 and Japanese Patent Application No. 2017-51354 filed on March 16, 2017 to the Japan Patent Office, and the entirety of these applications are incorporated by reference in this specification.

The present invention can be used in pharmaceuticals and their development.

Claims (41)

A compound of formula (I):

In the formula,
(a) R ¹ is heptadecanoyl and R ² is H, or (b) R ¹ is H and R ² is 5Z,8Z,11Z,14Z-icosatetraenoyl;
The following (1) to (5):
(1) ^R3 is PO(OH) ₂ , ^R4 is H, and ^R5 is H;
(2) ^R3 is H, ^R4 is H, and ^R5 is PO(OH) ₂ ;
(3) ^R3 is PO(OH) ₂ , ^R4 is PO(OH) ₂ , and ^R5 is H;
(4) ^R3 is PO(OH) ₂ , ^R4 is H, and ^R5 is PO(OH) ₂ ; or (5) ^R3 is PO(OH) ₂ , ^R4 is PO(OH) ₂ , and ^R5 is PO(OH) ₂ ;
Compound. A composition comprising the compound of claim 1. A compound of formula (II):

In the formula,
^R1 is heptadecanoyl and ^R2 is 5Z,8Z,11Z,14Z-icosatetraenoyl;
The following (1) to (5):
(1) ^R3 is PO(OH) ₂ , ^R4 is H, and ^R5 is H;
(2) ^R3 is H, ^R4 is H, and ^R5 is PO(OH) ₂ ;
(3) ^R3 is PO(OH) ₂ , ^R4 is PO(OH) ₂ , and ^R5 is H;
^2. A method for producing the compound according to claim 1 ^, comprising the step of deacylating the compound by contacting it with an alkylamine, wherein ( ₄ ) ^R3 is PO(OH) ₂ , ^R4 is _{H, and R5 is PO(OH)2} ^, _or ( ⁵ ) R3 is PO(OH)2, R4 is PO(OH)2, and R5 is PO(OH) ₂ . The method according to claim 3 , wherein the alkylamine is methylamine. The method according to claim 3, wherein the deacylation is carried out under milder conditions than the dediacylation conditions. The method according to claim 5, wherein the mild conditions are achieved by shortening the reaction time, reducing the concentration of the alkylamine, reducing the reaction temperature, or a combination thereof, among the dediacylation conditions. The method according to claim 5, wherein the mild conditions are: use of methylamine as the alkylamine; a reaction time of 10 minutes or less; a methylamine concentration of 11% or less; and a reaction temperature of 53°C or less; or a combination of the reaction time, the concentration, and the reaction temperature. (A) a compound of formula (II):

In the formula,
^R1 is heptadecanoyl and ^R2 is 5Z,8Z,11Z,14Z-icosatetraenoyl;
The following (1) to (5):
(1) ^R3 is PO(OH) ₂ , ^R4 is H, and ^R5 is H;
(2) ^R3 is H, ^R4 is H, and ^R5 is PO(OH) ₂ ;
(3) ^R3 is PO(OH) ₂ , ^R4 is PO(OH) ₂ , and ^R5 is H;
A method for producing a compound according _{to claim 1 having a desired acyl group at a desired position, comprising the steps of: (4) providing a compound in which R3 is PO(OH)2} ^{, R4} _is ^H , and ^R5 is PO ⁽ OH) ₂ ; or ( ⁵ ) R3 is PO(OH) ₂ , R4 is PO(OH)2, and R5 is PO(OH) ₂ ; and (B) deacylating the compound by contacting it with an alkylamine. concentrating the phospholipids by contacting the sample with an anion exchange resin;
protecting the phosphate group in the acidic phospholipid with a protecting group; and identifying or quantifying the compound according to claim 1 in the phospholipid by mass spectrometry.
2. A method for identifying or quantifying a compound according to claim 1, comprising: Further, the compound in the phospholipid is represented by the following formula (III):

In the formula,
R ¹ is C(═O)-R ^1′ , R ^1′ is an alkyl or alkenyl group, R ² is C(═O)-R ^2′ , R ^2′ is an alkyl or alkenyl group;
^R3 is H or PO(OH) ₂ ,
^R4 is H or PO(OH) ₂ ,
^R5 is H or PO(OH) ₂ ;
The method of claim 9, wherein the compound is also identified or quantified. The method of claim 9 or 10, wherein the protecting group comprises an alkyl group. The method of claim 9 or 10, wherein the protecting group comprises a methyl group. 11. The method of claim 9 or 10, wherein the mass spectrometry comprises selected reaction monitoring (SRM) with a triple quadrupole mass spectrometer. 14. The method of claim 13, further comprising identifying or quantifying the fatty acid side chains and/or phosphate groups of the compound of claim 1 using liquid column chromatography or ion mobility separation. The method of claim 9, further comprising identifying the location of the phosphate group. The method according to any one of claims 9 to 15, wherein in the mass spectrometry, acylglycerol is selected as a product ion (fragment ion) to identify or quantify the fatty acid side chain and/or phosphate group. A method for supporting cancer diagnosis without the involvement of medical professionals by the identification or quantification according to any one of claims 9 to 16. A method for measuring, detecting or identifying the compound according to claim 1 in a sample, the method comprising the steps of: A) subjecting the sample to liquid column chromatography-mass spectrometry (LC-MS) or ion mobility separation-mass spectrometry (IMS-MS); and B) identifying the position of a phosphate group of the compound according to claim 1 by the elution position of a peak in LC or IMS in the LC-MS or IMS-MS. 20. The method of claim 18, wherein step A) comprises subjecting the sample to chromatography. the mass spectrometry is liquid column chromatography-mass spectrometry (LC-MS);
The method according to claim 18, wherein the step B) of identifying is a step of identifying the position of the phosphate group of the compound according to claim 1 based on the elution position of a peak in LC by the LC-MS. The method according to any one of claims 18 to 20, wherein the type of acyl group contained in the compound according to claim 1 is also identified in the identifying step. The method according to any one of claims 18 to 21, characterized in that the measuring, detecting or identifying is carried out by distinguishing the compound according to claim 1 from other inositol-containing phospholipids. The method according to any one of claims 18 to 22, wherein the sample is prepared under conditions that do not cause decomposition of phosphatidylinositol phosphates and/or lysophosphatidylinositol phosphates. 24. The method of claim 23, wherein the conditions include the absence of a deacylation treatment. The method according to any one of claims 20 to 24, wherein the liquid column chromatography is selected from the group consisting of a chiral column, a reversed-phase column, and column chromatography that separates hydrophilic groups and hydrophobic groups. The method according to any one of claims 18 to 25, wherein the liquid column chromatography is combined with ion mobility separation. The method according to any one of claims 18 to 26, further comprising the step of quantifying the compound according to claim 1. The method of any one of claims 18 to 27, wherein the sample is an intact sample. The method according to any one of claims 18 to 28, wherein the identification is carried out without labelling the sample. The mass spectrometry is ion mobility separation-mass spectrometry (IMS-MS),
The method according to claim 18, wherein the step B) of identifying is a step of identifying the position of the phosphate group of the compound according to claim 1 based on the elution position of a peak in IMS-MS. Method according to claim 30, comprising any one or more of the features of claims 21 to 25, 27 to 29. An apparatus for measuring, detecting or identifying the positions of acyl groups and phosphate groups of the compound according to claim 1 in a sample, the apparatus comprising: a mass spectrometry (MS) which is liquid column chromatography-mass spectrometry (LC-MS) or ion mobility separation-mass spectrometry (IMS-MS); and an application section which applies the sample to the MS under conditions which do not decompose phosphatidylinositol phosphate. 33. The apparatus of claim 32, further comprising a device for chromatography. The apparatus of claim 32, wherein the mass spectrometry is liquid column chromatography-mass spectrometry (LC-MS), and the application section is an application section that applies the sample to the LC-MS under conditions that do not cause decomposition of phosphatidylinositol phosphates. The apparatus according to claim 34, wherein the liquid column chromatography is selected from the group consisting of all column chromatography that separates hydrophilic and hydrophobic groups, such as chiral columns and reversed-phase columns. 36. The device of any one of claims 32 to 35, further comprising means for detecting or quantifying the compound of claim 1. The apparatus of claim 32, wherein the mass spectrometry is ion mobility separation-mass spectrometry (IMS-MS), and the application unit is an application unit that applies the sample to the IMS-MS under conditions that do not decompose phosphatidylinositol phosphates. A method for separating or purifying the compound according to claim 1 in a sample according to the position of a phosphate group while retaining an acyl group, the method comprising the steps of: A) applying the sample to liquid column chromatography-mass spectrometry (LC-MS) or ion mobility separation-mass spectrometry (IMS-MS); and B) collecting the compound according to claim 1 having a phosphate group at a desired position specified by the elution position of a peak from the LC or IMS eluate in the LC-MS or IMS-MS. A method for producing a sample containing the compound according to claim 1 having phosphate groups at positions that are fewer in number than the number of positions of the phosphate groups of the compound according to claim 1 contained in a mixture in which two or more phosphate groups are present at the positions of the compound according to claim 1, comprising the steps of:
A) subjecting the sample to liquid column chromatography-mass spectrometry (LC-MS) or ion mobility separation-mass spectrometry (IMS-MS); and B) collecting a fraction containing the compound according to claim 1 having a phosphate group at a position of interest identified by the elution position of a peak from the LC or IMS eluate in the LC-MS or IMS-MS. The method of claim 39, wherein the number of types of phosphate group positions of the compound described in claim 1 contained in the mixture is one type, which is less than the number of types of phosphate group positions of the compound described in claim 1. A method for producing a compound according to claim 1 or a composition comprising the compound, comprising the steps of:
A) subjecting a mixture of the compound according to claim 1 having two or more phosphate group positions to liquid column chromatography-mass spectrometry (LC-MS) or ion mobility separation-mass spectrometry (IMS-MS); and B) collecting a fraction containing the compound according to claim 1 having a phosphate group at a position of interest specified by the elution position of the peak from the LC or IMS eluate in the LC-MS or IMS-MS.