CN106814128B - Method for detecting exosome small-molecule metabolite by using mass spectrum - Google Patents
Method for detecting exosome small-molecule metabolite by using mass spectrum Download PDFInfo
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Abstract
The invention discloses a method for detecting exosome small-molecule metabolites by using mass spectrometry, which comprises the following steps: preparing instruments and reagents; preparing a matrix of iron oxide-containing nanoparticles; preparing exosome samples, respectively sampling the exosome samples diluted in different proportions on a target plate, and drying at room temperature; dropping iron oxide-containing nanoparticle matrix on the exosome sample, and drying at room temperature; and carrying out mass spectrum detection and analyzing the mass spectrum detection result to obtain a conclusion. The method adopts micro-nano particle materials as a matrix, and can quickly and efficiently use a very small amount of biological samples to realize qualitative and quantitative detection of molecules to be detected in mass spectrometry; meanwhile, background interference and hot spot effects of the traditional matrix can be removed, and the detection of metabolic molecules in the exosome sample is realized.
Description
Technical Field
The invention relates to the field of mass spectrometry detection, in particular to a method for detecting exosome small-molecule metabolites by matrix-assisted laser desorption ionization mass spectrometry.
Background
Exosomes are small vesicles with diameters of about 30-150nm and densities of 1.13-1.21g/m 1. Exosomes are naturally present in body fluids, including blood, saliva, urine, and breast milk, and are membrane vesicles secreted by living cells from late endosomes (also known as multivesicular bodies). Exosomes have many important functions, such as performing protein trafficking functions, specifically targeting receptor cells, exchanging proteins and lipids or triggering downstream signaling events. Exosomes also transport nucleic acids, and participate in cell-cell communication. In conclusion, the protein, RNA and fat components of the polypeptide are specific, carry some important metabolic small molecules and are expected to play a role in early diagnosis of various diseases. At present, the analysis method of exosome mainly relates to protein, polypeptide and gene, and the detection and analysis of metabolic molecule still has a great research gap. Traditional analysis methods such as biochemical methods and electrospray ionization mass spectrometry (ESI) are difficult to realize fast and efficient detection of small molecules due to their complicated sample pretreatment methods. Meanwhile, the currently commonly used method usually requires a sample amount in the milliliter level for a single detection, which brings great obstacles to the detection of small molecules. Therefore, matrix-assisted laser desorption/ionization (MALDI) is adopted as a novel analysis mode for soft ionization biological samples, and substances with different molecular weight grades can be analyzed. It represents a simple, fast, accurate way to detect multiple biomolecules simultaneously. The effectiveness of this method is justified not only in theory but also in practical applications. However, the conventional matrix is easy to generate background noise at the small molecular weight end (m/z <1000), and brings great interference to the detection of the small molecules. And in practical biological systems, biological samples are often quite complex. The existence of various biological macromolecules, different pH values and different salt contents can bring obstruction to the detection of small molecules. Therefore, the traditional matrix is difficult to meet the requirement of small molecule detection, and a novel matrix material which can be used for biological system detection and has certain anti-interference and certain salt tolerance is in urgent need of development.
Disclosure of Invention
In view of the above-mentioned drawbacks of the prior art, the present invention provides a novel particle-assisted mass spectrometry based detection technique. By adopting the micro-nano particle material as the matrix, the defect of the traditional matrix is overcome, and the molecules to be detected in the exosome can be quantified quickly, with high flux and high sensitivity.
The technical scheme of the invention is as follows:
the invention provides a method for detecting an exosome small-molecule metabolite by using mass spectrometry, which comprises the following steps:
step 1: preparation of instruments and reagents: laser desorption ionization mass spectrometry and positive ion mode detection, wherein only mass spectrum signals with the signal-to-noise ratio larger than 10 are used for qualitative and quantitative analysis;
step 2: preparing an iron oxide-containing nanoparticle matrix, the preparing of the iron oxide-containing nanoparticle matrix comprising the steps of:
2.1: dissolving ferric trichloride and trisodium citrate in an ethylene glycol solution;
2.2: adding sodium acetate into the mixed solution, and performing ultrasonic treatment at room temperature for half an hour until the solution becomes a homogeneous system;
2.3: the reaction is carried out in a Teflon high-pressure reaction kettle and is carried out for more than 8 hours at the temperature of 150-;
2.4: repeatedly washing the iron-containing oxide nanoparticles obtained in the step 2.3 with ethanol and deionized water, and finally drying at 62-70 ℃ for later use;
2.5: resuspending the iron oxide-containing nanoparticles in deionized water for use as a matrix;
and step 3: extracting exosome, spotting the diluted exosome sample on a target plate, and drying at room temperature;
and 4, step 4: dropping iron oxide-containing nanoparticle matrix on the exosome sample, and drying at room temperature;
and 5: performing mass spectrometry detection on the exosome sample obtained in the step 4;
step 6: and analyzing the mass spectrum detection result to obtain a conclusion.
Further, the iron oxide-containing nanoparticle matrix has a diameter of less than 1 μm, a uniform particle size, and a rough surface.
Further, the iron oxide-containing nanoparticle matrix is FexOyOr a mixture thereof, wherein x is 10 or less and y is 0 or more and 10 or less.
Further, the size of the iron oxide-containing nanoparticle matrix ranges from 200nm to 300 nm.
Further, the rough surface of the iron oxide-containing nanoparticle matrix is composed of nano-spheres of 50nm or less. In terms of performance, the iron oxide-containing particles have ultraviolet absorption.
Further, the rough surface of the iron oxide-containing nanoparticle matrix is composed of nanospheres having a diameter of 5nm to 8 nm.
Further, the dilution factor of the exosome is less than 10000 times to obtain an exosome sample.
Further, exosomes include exosomes extracted from serum, plasma and cells.
Further, the detection molecular weight range is 10000Da or less.
Further, the molecules tested included sugars and amino acids.
The invention has the beneficial effects that: the micro-nano iron-containing oxide particle matrix has low preparation cost, can be manufactured in a large scale and has simple synthesis steps; the micro-nano iron oxide particle matrix can remove background interference and hot spot effect of the traditional matrix and has high salt tolerance. According to the invention, trace exosomes are only consumed, and metabolites in the exosomes can be rapidly, efficiently and quantitatively detected and analyzed on the premise of no enrichment and separation operation; the whole detection process has simple steps, low cost and high flux; the obtained quantitative result has high accuracy and can be applied to clinic.
The present invention will be further described with reference to the accompanying drawings to fully illustrate the objects, technical features and technical effects of the present invention.
Drawings
Fig. 1 is a representation picture of iron oxide particles prepared in a preferred embodiment of the present invention, fig. 1a is an SEM representation picture, and fig. 1b is a TEM representation picture;
FIG. 2 is a mass spectrum of the method for detecting metabolic small molecules in exosomes extracted from serum by the laser desorption ionization technique in the specific example 1. A-up represents urea and a-up represents urea,
represents lactic acid, ● represents cysteine, ■ represents mannitol;
FIG. 3 is a mass spectrum of the method for detecting metabolic small molecules in exosomes extracted from cells by the laser desorption ionization technique in the embodiment 2. A-up represents urea and a-up represents urea,
which represents a lactic acid, is a lactic acid,
shown is a valine, which is shown in the formula,
the amino acid is shown as arginine,
showing glucose.
Detailed Description
The invention is further described below with reference to the drawings and the embodiments.
The preparation of the iron oxide-containing nanoparticle matrix comprises the following steps:
step 1: dissolving ferric trichloride and trisodium citrate in an ethylene glycol solution;
step 2: adding sodium acetate into the mixed solution, and performing ultrasonic treatment at room temperature for half an hour until the solution becomes a homogeneous system;
and step 3: the reaction is carried out in a Teflon high-pressure reaction kettle and is carried out for more than 8 hours at the temperature of 150-;
and 4, step 4: repeatedly washing the iron-containing oxide nanoparticles obtained in the step (3) with ethanol and deionized water, and finally drying at 62-70 ℃ for later use;
and 5: the iron oxide-containing nanoparticles were resuspended in deionized water and used as a matrix.
Characterization of the matrix:
the instruments used for characterization were: the instruments used for characterization were: adopting a JEOL JEM-2100F instrument to obtain a transmission electron microscope picture, a high-resolution transmission electron microscope picture and a selected area electron diffraction pattern; preparing a scanning electron microscope sample by using a silicon wafer, and obtaining a scanning electron microscope picture by using a Hitachi S-4800 instrument;
the characterization result is as follows:
the resulting particulate material was spherical and had a size of about 250 nm. As shown in fig. 1, it can be seen from the transmission electron microscope picture that the particulate material has a subunit structure, and it can also be seen from the high resolution transmission electron microscope picture obtained by selecting the edge region of the particulate material, and the regular lattice pattern further proves that the particulate material is composed of a plurality of nanocrystals. According to the measured scanning electron microscope picture, the surface of the particle material is rough and uniform in size. The structure is an important condition for becoming a good matrix material for biomolecule mass spectrometry detection.
The application of matrix-assisted laser desorption ionization mass spectrometry in the detection and analysis of secreted body metabolic molecules is further illustrated by several exemplary application examples.
Example 1: detection of metabolic small molecules in exosomes extracted from serum
(1) Preparation of instruments and reagents: the laser analysis ionization mass spectrometer only uses mass spectrum signals with signal-to-noise ratio larger than 10 for separationAnd (6) analyzing. Using AB SCIEX TOF/TOFTM5800 Mass spectrometer, Nd: YAG laser, wavelength 355 nm. And the working mode of pulse electric field delay extraction and reflection and the positive ion mode are adopted for detection. Data were observed, processed, and analyzed using a DataExplorer, and only mass spectra signals with a signal-to-noise ratio greater than 10 were used for analysis.
(2) Iron oxide-containing nanoparticles are prepared.
(3) Taking 1-10 ml serum, carrying out ultra-high speed centrifugation at the rotating speed of 15000-25000G for 4-10 hours to extract exosome, and preparing exosome solution with the concentration of 0.1-10 mg/ml.
(4) The MALDI target plate was cleaned with formic acid, absolute ethanol, deionized water sequentially and ultrasonically for 1.5 hours.
(5) And (3) ultrasonically oscillating and dispersing the iron-containing oxide nanoparticles in deionized water, mixing with an exosome solution, and spotting on a dry MALDI target plate.
(6) After the sample is dried, the sample is analyzed by laser and ionized mass spectrometry is used for detection.
The results are shown in FIG. 2.
Example 2: detection of metabolic small molecules in exosomes extracted from cells
(1) Preparation of instruments and reagents: the laser desorption ionization mass spectrometer only uses mass spectrum signals with signal-to-noise ratio larger than 10 for analysis. Using AB SCIEX TOF/TOFTM5800 Mass spectrometer, Nd: YAG laser, wavelength 355 nm. And the working mode of pulse electric field delay extraction and reflection and the positive ion mode are adopted for detection. Data were observed, processed, and analyzed using a DataExplorer, and only mass spectra signals with a signal-to-noise ratio greater than 10 were used for analysis.
(2) Iron oxide-containing nanoparticles are prepared.
(3) Taking 1ml-10ml of cell solution (applicable to various types of cells), carrying out ultra-high speed centrifugation at the rotating speed of 15000-25000G for 4-10 hours to extract exosomes, and preparing the exosome solution with the concentration of 0.1 mg/ml-10 mg/ml.
(4) The MALDI target plate was cleaned with formic acid, absolute ethanol, deionized water sequentially and ultrasonically for 1.5 hours.
(5) And (3) ultrasonically oscillating and dispersing the iron-containing oxide nanoparticles in deionized water, mixing with an exosome solution, and spotting on a dry MALDI target plate.
(6) After the sample is dried, the sample is analyzed by laser and ionized mass spectrometry is used for detection.
The results are shown in FIG. 3.
The foregoing detailed description of the preferred embodiments of the invention has been presented. It should be understood that numerous modifications and variations could be devised by those skilled in the art in light of the present teachings without departing from the inventive concepts. Therefore, the technical solutions available to those skilled in the art through logic analysis, reasoning and limited experiments based on the prior art according to the concept of the present invention should be within the scope of protection defined by the claims.
Claims (6)
1. A method for detecting exosome small-molecule metabolites using mass spectrometry, comprising the steps of:
step 1: preparation of instruments and reagents: laser desorption ionization mass spectrometry and positive ion mode detection, wherein only mass spectrum signals with the signal-to-noise ratio larger than 10 are used for qualitative and quantitative analysis;
step 2: preparing an iron oxide-containing nanoparticle matrix, the preparing of the iron oxide-containing nanoparticle matrix comprising the steps of:
2.1: dissolving ferric trichloride and trisodium citrate in an ethylene glycol solution;
2.2: adding sodium acetate into the mixed solution, and performing ultrasonic treatment at room temperature for half an hour until the solution becomes a homogeneous system;
2.3: the reaction is carried out in a Teflon high-pressure reaction kettle and is carried out for more than 8 hours at the temperature of 150-;
2.4: repeatedly washing the iron oxide-containing nanoparticles obtained in the step 2.3 with ethanol and deionized water, and finally drying at 62-70 ℃ for later use;
2.5: resuspending the iron oxide-containing nanoparticles in deionized water for use as a matrix;
and step 3: extracting exosome, spotting the diluted exosome sample on a target plate, and drying at room temperature;
and 4, step 4: spotting the iron oxide-containing nanoparticle matrix on the exosome sample, drying at room temperature;
and 5: performing mass spectrometry detection on the exosome sample obtained in the step 4;
step 6: analyzing the mass spectrum detection result to obtain a conclusion;
wherein the detected molecules comprise saccharides and amino acids, and the detected molecular weight range is less than or equal to 10000D;
the iron oxide-containing nanoparticle matrix has a diameter of less than 1 μm and a uniform particle size, and has a rough surface;
the iron oxide-containing nanoparticle matrix is FexOyOr a mixture thereof, wherein x is 10 or less and y is 0 or more and 10 or less.
2. The method for detecting exosome small-molecule metabolites according to claim 1, wherein the iron-containing oxide nanoparticle matrix is in the size range of 200 nm-300 nm.
3. The method for detecting exosome small-molecule metabolites according to claim 1, wherein the rough surface of the iron-oxide-containing nanoparticle matrix consists of nanoballs below 50 nm.
4. The method for detecting exosome small-molecule metabolites according to claim 1, wherein the rough surface of the iron-oxide-containing nanoparticle matrix is composed of nanoballs with a diameter of 5nm to 8 nm.
5. The method for detecting exosome small-molecule metabolites according to claim 1, wherein the exosome is diluted by less than 10000 times to obtain the exosome sample.
6. The method for detecting exosome small-molecule metabolites according to claim 1, wherein the exosomes comprise exosomes extracted from serum, plasma and cells.
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| CN107643337B (en) * | 2017-09-18 | 2020-08-18 | 浙江亿纳谱生命科技有限公司 | Matrix, preparation method thereof and biological sample detection method |
| CN107356662B (en) * | 2017-09-19 | 2020-08-18 | 浙江亿纳谱生命科技有限公司 | Matrix material, preparation method and application thereof, and biological sample detection method |
| CN109444251B (en) * | 2018-11-23 | 2021-12-21 | 亿纳谱（浙江）生物科技有限公司 | Application of nano matrix in nucleic acid detection |
| CN111413396A (en) * | 2020-04-21 | 2020-07-14 | 四川大学 | Application of ferric oxide nano material and MA L DI-TOF MS in detection of small molecule metabolites |
| CN111413395A (en) * | 2020-04-21 | 2020-07-14 | 四川大学 | Application of porous silicon nanowires combined with MALDI-TOF MS in the detection of metabolic small molecules |
| CN113447560B (en) * | 2021-05-24 | 2022-12-09 | 上海交通大学 | Small molecule metabolite fragmentation control method based on metal ion addition and application |
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