CN107085032A - A Peptide Derivatization Method and Its Application in MALDI‑TOF‑MS Detection of Drug Metabolites - Google Patents

Abstract

The invention discloses an analysis method of chemical derivatization matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF-MS). The present invention first uses dialdehyde compounds to modify drug metabolites containing amino structures, so that drug metabolites can derive groups with aldehyde groups, and then use polypeptides to derivatize the aldehyde derivatives, and then use MALDI‑TOF ‑MS detection. After peptidification of drug metabolites, simple and single quasi-molecular ion peaks can be obtained, which not only significantly improves the identification of these metabolites in mass spectrometry, but also greatly improves the detection sensitivity of drug metabolites in mass spectrometry. The method has high sensitivity, wide application range, convenient operation, minimal matrix interference, and low sample requirement. It can quantitatively detect amino metabolites with extremely low content in metabolites without preparing attached matrix and derivatization reagents. It has great application prospects in metabolomics research.

Description

A Peptide Derivatization Method and Its Application in MALDI-TOF-MS Detection of Drug Metabolites Applications

technical field

The invention relates to the field of mass spectrometry detection, in particular to the method of utilizing polypeptide derivatized metabolites combined with MALDI-TOF-MS and its application in the field of metabolomics.

Background technique

Metabolomics is another research field that characterizes the status of living systems after genomics and proteomics. The detection of metabolites in organisms has profound significance for the study of gene expression, exploration of life processes, diagnosis of diseases, and prevention of diseases. . Therefore, the analysis of metabolites is an important direction in the field of analytical chemistry, and even in the field of life sciences.

Mass spectrometry, as an important method for the study of small molecule metabolites, has significant advantages such as high sensitivity, good specificity, and strong qualitative ability. Currently, the most common mass spectrometric detection technique for small molecule metabolites is liquid chromatography-mass spectrometry. Although the sensitivity of LC-ESI-MS is high, the detection time of LC-MS is long, which is not suitable for rapid high-throughput analysis.

Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF-MS) is widely used in the detection of macromolecular samples such as DNA, proteins, peptides, and polysaccharides due to its rapid, simple, and high-throughput characteristics. Considering the advantages of MALDI-TOF-MS detection such as rapidity, simplicity, high sensitivity, and high throughput, we tried to extend it to the detection of small molecule metabolites. Although MALDI-DOF-MS has significant advantages in the detection of macromolecular substances, it is not suitable for the detection of small molecular substances such as metabolites, mainly because of the commonly used matrix CHCA, DHB, 3-HPA, 2,4, 6-THP etc. will have serious matrix interference to the target analyte in the small molecule (<500Da) segment. In order to overcome this shortcoming, the methods reported in the existing literature are mainly divided into two categories: 1) the matrix with little matrix interference is adopted, such as porous silicon, nano-microspheres, metal oxides, carbon materials, etc., but there is still no convenient, It is widely applicable to the development of matrices; 2) Design macromolecular derivatization reagents to derivate small molecule analytes. The preparation of derivatization reagents reported in the existing literature is usually very cumbersome and has certain limitations. Therefore, it is urgent to develop a simple, highly sensitive and universal derivatization method in the analysis of small molecule chemicals by MALDI-TOF-MS.

Aiming at the above problems, the present invention firstly develops a novel MALDI-TOF-MS detection method using polypeptide as a derivatization reagent to derivatize small molecule metabolites, thereby overcoming the advantages of MALDI-TOF-MA in the detection of small molecule substances and developing MALDI-TOF - Application of MS in the field of metabolomics detection. It has the advantages of simple and fast operation, less sample consumption, etc., and is especially suitable for the analysis of biological samples.

Contents of the invention

The technical problem to be solved by the present invention is to provide a highly sensitive and universal MALDI-TOF-MS analysis method that can be applied in the field of detection of small molecule metabolites and combined with chemical derivatization strategies;

The derivation technology and analysis scheme provided by the present invention comprise the following steps:

(1) After molecular simulation and calculation of the polypeptide by the PM3 method, the polypeptide used for derivatization is screened, and the polypeptide contains groups such as arginine and phenylalanine that are easily ionized in the MALDI source;

(2) The polypeptide obtained in step (1) is used to derivate small molecular substances to be tested, so that these small molecules are equipped with easily ionized amino acid residue chains.

The present invention derivatizes amino group-containing compounds through dialdehyde, and then screens high-sensitivity polypeptides as derivatization reagents through molecular simulation, and uses N-terminal modified cysteine and maleimide polypeptides to derivate aldehyde compounds respectively. Then use MALDI-TOF-MS to detect these peptide-labeled small molecule metabolites.

A derivatization method: the polypeptide P2 whose sequence is CFRGLRGF and the polypeptide P7 whose sequence is KFRGLRGF (modified maleimide on Lys) are used as derivatization reagents of aldehyde substances, water is used as a reaction solvent, and phosphate buffer solution is used as a buffer agent;

The reaction conditions are: aldehyde compound/polypeptide P2 (20-100, c/c) is added to a phosphate buffer solution with a pH of 5-7, and derivatized at 4-37°C for 0.2-2h;

The derivatization reaction refers to the dehydration condensation reaction between the Cys-containing polypeptide and the aldehyde compound.

Preferably, the aldehyde compound and the polypeptide P2 are added to a phosphate buffer solution with a pH of 4 at a molar ratio of 50:1, and a dehydration condensation reaction occurs at 25° C. for 5 hours.

Since the polypeptide derivatization reagent has amino acid residues that are easily ionized in combination with protons in MALDI-MS, including Arg (arginine), Phe (phenylalanine), etc., it is found that the screened polypeptide has a higher proton affinity, so that it is easy to bind protons in mass spectrometry and has high sensitivity.

A quantitative analysis method of peptide derivatization combined with MALDI-TOF-MS: After derivatizing the small molecule analyte, mix it with the corresponding stable isotope internal standard 1:1, mix 1 μL matrix (CHCA) with 1 μL test solution Spot the plate; after drying at room temperature, enter the matrix-assisted laser desorption ionization-time-of-flight mass spectrometry analysis, and perform quantitative analysis according to the ratio of the peak intensity of the target analyte spectrum to the peak intensity of the stable isotope spectrum.

Application of the described polypeptide derivatization method combined with MALDI-TOF-MS analysis method in the field of metabolomics analysis: the polypeptides screened for derivatization contain Arg (arginine), Phe, which are easily ionized in MALDI sources (Phenylalanine) group, after labeling the small molecules to be tested, these small molecules can be brought into the easily ionized amino acid residue chain, which significantly enhances their ionization efficiency in MALDI-TOF-MS, thus leading to the method’s Sensitivity is greatly improved. Since the minimum detection limit (LOD) of peptides in MALDI-TOF-MS can reach fmol, the LOD of peptide-derived small molecule analytes in mass spectrometry can also reach fmol levels. Moreover, this peptide derivatization strategy can be applied to the detection of many other small molecule chemicals. In addition to the aldehyde compounds described in the invention, it can also be extended to small molecules such as ketones, carboxylic acids, alcohols and phenols, and esters. In the MALDI-TOF-MS analysis of compounds, the detection of these small molecular compounds is of great significance to the research of metabolomics.

The beneficial effects of the present invention are as follows:

1. In the present invention, the method is easy to operate, and the analyte is directly analyzed by MALDI-TOF-MS after labeling.

2. The peptide derivation method designed in the present invention can significantly improve the detection sensitivity of small molecule analytes in MALDI-TOF-MS (the sensitivity is increased by 50,000 times compared with that before derivation), and the detection limit can reach the fmol level.

3. The polypeptide derivatization reagent used in the present invention makes the small molecule to be tested bear a marker with a large molecular weight, which avoids the interference of the matrix effect of the low molecular segment (<500Da) in MALDI-TOF-MS on the detection.

4. The polypeptides and isotopic polypeptides involved in the present invention can be purchased as commercial products and used for quantitative analysis.

5. The derivatization reaction designed in the present invention has high universality. Since there are many groups available for modification on the polypeptide of the derivatization reagent, the analysis of various small molecular compounds has a high application prospect.

detailed description

1. Configuration of the working solution: Dissolve the derivatized polypeptide in water to prepare a 100 μM solution for later use, and dissolve the aldehyde derivative of doxorubicin metabolite to be tested in parallel methanol to prepare a stock solution.

2. Preparation of aldehyde metabolites. Dissolve the metabolites and dialdehyde compounds in alcohol solvents at a molar ratio of 1:1.2, add 1 drop of glacial acetic acid, stir the reaction at 50°C, and spin dry the solvent spare.

3. The method of polypeptide derivatization is as follows: dissolve the test substance of the polypeptide derivatization kit in aqueous solution or other solvents, and add it into a certain pH phosphate buffer according to an appropriate proportion, so that the concentration of the test substance in the reaction system The concentration was 10 μM, and the derivatization reaction was carried out at an appropriate temperature after vortex mixing. Derivatization conditions are aldehyde compounds: polypeptide P2 (20-100, c/c) is added to a phosphate buffer solution with a pH of 4-6, and derivatized at 25-37° C. for 1-10 hours.

4. After the derivatization reaction is completed, mix the product solution with the stable isotope internal standard solution, vortex and mix, and mix 1 μL matrix (CHCA) according to 1 μL sample to spot the plate; after air-drying at room temperature, enter matrix-assisted laser desorption ionization-time-of-flight mass spectrometry , analyzed in positive ion reflectance mode.

5. The method for preparing the stable isotope internal standard described in step 4 is to derivatize the stable isotope polypeptide P2 (N and 6 C marks on the polypeptide leucine) with an excess of aldehyde compound, and spin dry in vacuum , to remove excess small molecules, and then redissolve in water title internal standard solution.

6. The mass spectrometry detection condition described in step 4 is: Matrix-Assisted Desorption Ionization-Time-of-Flight Mass Spectrometer (MALDI-TOF-MS) of Waters Company. MALDI-MS analysis uses a nitrogen laser with a wavelength of 337nm, a pulse width of 3ns, an accelerating voltage of 20kV, and detection in positive ion reflection mode. Each spectrum is the average of 200 laser scans.

Example 1 Determination of detection limit of doxorubicin after derivatization with aldehyde group and polypeptide

Dissolve 1mmol of doxorubicin in 5mL of methanol, add 1.1mmol of glyoxal, add a drop of glacial acetic acid to catalyze the reaction, stir the reaction at 50°C for 3 hours, monitor the progress of the reaction by thin-layer chromatography, and remove the solvent under reduced pressure after the reaction is complete. Schiff's base spare. Dissolve the test substance of the peptide derivatization kit in an aqueous solution, add it to a certain pH phosphate buffer, make the concentration of the test substance in the reaction system 10μM, vortex and mix and carry out the derivatization reaction at a suitable temperature . Derivatization conditions are aldehyde compounds: polypeptide P2 (20-100, c/c) is added to a phosphate buffer solution with a pH of 4-6, and derivatized at 25-37° C. for 1-10 hours. Spot the plate with 1 μL of matrix; after drying at room temperature, enter matrix-assisted laser desorption ionization-time-of-flight mass spectrometry analysis to obtain the detection limit of doxorubicin.

The determination of the content of metabolites of doxorubicin in embodiment 2 MCF-7 cells

After the pre-dosed MCF-7 cells were crushed, doxorubicin metabolites were extracted, 1 μL of doxorubicin metabolite extract was dissolved in 5 mL of methanol, 1.1 μL of malondialdehyde was added, and a drop of glacial acetic acid was added to catalyze the reaction, 50 The reaction was stirred at ℃ for 3 hours, and the progress of the reaction was monitored by thin-layer chromatography. After the reaction was complete, the solvent was removed under reduced pressure to obtain the Schiff base for future use. Dissolve the test substance of the peptide derivatization kit in an aqueous solution, add it to a certain pH phosphate buffer, make the concentration of the test substance in the reaction system 10μM, vortex and mix and carry out the derivatization reaction at a suitable temperature . Derivatization conditions are aldehyde compounds: polypeptide P2 (20-100, c/c) is added to a phosphate buffer solution with a pH of 4-6, and derivatized at 25-37° C. for 1-10 hours. Stable isotope internal standard was added to make the concentration 1 μM. After mixing, 1 μL of matrix (CHCA) was taken to spot the plate. After air-drying at room temperature, it was put into matrix-assisted laser desorption ionization time-of-flight mass spectrometry, and positive ion reflectance mode was used for analysis. According to the target analyte’s spectrum The ratio of line intensity to stable isotope spectrum line intensity was quantitatively analyzed, and finally the content of doxorubicinol in MCF-7 cells added for 3 hours was 2.3 μM.

Claims (5)

1. a kind of polypeptide derives with the method for changing amino metabolism structure micromolecular compound combination MALDI-TOF-MS, its feature Comprise the following steps：

(1) micromolecular compound：Using twain-aldehyde compound compound as linking arm, modified to amino-compound, made by condensation reaction Drug metabolite structure derives aldehyde groups.

(2) many derived chemotactic peptide micromolecular compounds：By aldehyde compound, polypeptide with mol ratio 50-100:1 is added to pH for 4-6 Phosphate buffer in, condensation reaction occurs at 5-37 DEG C, the amino acid sequence N-terminal cysteine modified of polypeptide is used as aldehyde Class compound derivatization reagent.

(3) MALDI-TOF-MS is analyzed：Analyte derivative is according to 1 μ L sample mixed-matrix alpha-cyano -4- hydroxycinnamic acids CHCA Point plate, room temperature carries out Matrix Assisted Laser Desorption lonization-Time of Flight after air-drying, using cation reflective-mode.

2. aldehyde radical derivatization method according to claim 1 is characterized in that, twain-aldehyde compound chemical combination is selected from glyoxal, the third two Aldehyde, butanedial, glutaraldehyde, hexandial, dialdehyde in heptan, suberic aldehyde.The reaction dissolvent of twain-aldehyde compound compound and metabolite is selected from first Alcohol, ethanol, isopropanol, n-butanol, the tert-butyl alcohol, the reaction time are 0.5-5h, and reaction temperature is 20-100 DEG C.

3. polypeptide derivatization method according to claim 1, it is characterised in that aldehyde derivative in step (1) with it is many The mol ratio of peptide is 50-100:1 is added in the phosphate buffer that pH is 4-6, occurs condensation reaction, the reaction time is 0.5- 5h。

4. a kind of polypeptide derives with the analysis method for changing amino metabolism structure micromolecular compound combination MALDI-TOF-MS, its It is characterised by, after small molecule determinand is combined according to 1-3 method aldehyde radical derivative and polypeptide, according to 1 μ L sample mixed-matrixes Alpha-cyano -4- hydroxycinnamic acids CHCA point plates, room temperature carries out Matrix Assisted Laser Desorption lonization-Time of Flight after air-drying and determined Property analysis.Can directly can be with corresponding stable isotope 1：1 mixes, according to 1 μ L sample mixed-matrix alpha-cyano -4- hydroxyl meat Cinnamic acid CHCA point plates, room temperature carries out Matrix Assisted Laser Desorption lonization-Time of Flight analysis after air-drying, according to target analysis The ratio of material spectral strength and stable isotope spectral strength carries out quantitative analysis to it.

5. the application in method described in any one of claim 1-3 metabonomic analysis field again.