Mass spectrometric detection pretreatment method for low-molecular-weight protein in plasma

Abstract

The invention provides a mass spectrometric detection pretreatment method for low-molecular-weight proteins in plasma, which comprises the following steps: combining 60% acetonitrile, 80% acetonitrile and 10% TCA (with or without DTT), removing high-abundance proteins in a plasma sample in a precipitate, and extracting the low-molecular-weight proteins in a supernatant so as to achieve the purpose of separation, so that the possibility of finding clinical disease markers is greatly improved. The method comprises the following steps: adding 60% and 80% acetonitrile and 10% TCA into a plasma sample, and precipitating high-abundance proteins such as albumin, globulin, lipoprotein and complement in the plasma; the low-molecular-weight proteins in the blood plasma supernatant precipitated by the method are subjected to different combinations, then the sample is subjected to chromatographic fractionation through trans-liquid chromatography under the alkaline pH condition, and finally mass spectrometry detection is performed by using a liquid chromatograph-mass spectrometer, so that the detection quantity of the low-molecular-weight proteins in the blood plasma sample is increased. The combination of these extracts increases the coverage of plasma low molecular weight proteomes.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of plasma low molecular weight protein mass spectrometry, and relates to a mass spectrometry pretreatment method for low molecular weight proteins in plasma BACKGROUND

[0002] Low molecular weight (LMW) proteins, including small proteins, protein fragments and peptides, can reflect the physiological homeostasis of the body or directly participate in the occurrence of diseases. Plasma or serum low molecular weight proteomics or peptidomics has always been a focus in the research field. Extracting low molecular weight proteins or peptides from plasma and then performing mass spectrometry is a typical method for plasma or serum low molecular weight proteomics or peptidomics research. However, peptidomics has significant diversity, and peptides can differ in size, polarity and solubility. An ideal peptidomics extraction method must have sufficient versatility to capture as many peptides as possible while removing high-abundance interfering proteins. Proteins in clinical samples are much larger than peptides, and the abundance of many proteins is also higher than that of peptides, and other components such as lipids often have a masking effect, which interferes with the separation and detection of peptides.

[0003] A commonly used method for peptide enrichment is centrifugal ultrafiltration, which uses a molecular weight cut-off (MWCO) membrane to separate low molecular weight and high molecular weight peptides. This method is fast, easy to operate, and can identify many unique peptides in serum and plasma. However, peptides with molecular weights close to the cut-off point may be lost, such as apolipoproteins, and some "sticky" lipophilic peptides may be lost due to binding to the membrane. In addition, most of the peptide content is lost when using a centrifugal MWCO device, while a simple precipitation method has an advantage in recovering low molecular weight components of plasma.

[0004] Adding an organic solvent will precipitate large molecular proteins, leaving low molecular weight proteins, peptides and other small molecules in the solution, which is a common extraction method for small molecule analysis. Organic precipitation has been used to detect low-abundance peptides in plasma samples. Although this method is very versatile, the composition of the precipitation solvent should be considered and verified for each matrix to ensure optimal recovery of peptides. Although the organic solvent precipitation method is very effective in removing high-abundance proteins, lipids and smaller analytes will still remain in the supernatant, and sometimes additional steps are needed to remove these analytes.

[0005] An alternative to the organic precipitation method is the acid extraction technique, for example, adding trichloroacetic acid is sufficient to precipitate high-abundance proteins in plasma. One important advantage of using a precipitation method is that the interaction of proteins with solvents / acid is rapid, so once the acid is added to the plasma, degradation associated with peptidases is immediately prevented.

[0006] Immunoaffinity peptide enrichment is an effective method to improve detection sensitivity, which is essential for detecting low levels of peptides. This method has been successfully applied to multiplexed quantitative analysis of gut hormones by using mixed antibody-coupled magnetic beads during extraction. Although this method is useful for quantifying targeted peptides, it is limited by the availability of suitable antibodies against peptides and their high cost. In addition, the recovery rate of peptides can be low, which means that this method requires a large amount of plasma, which may not be available.

[0007] Therefore, we attempt to combine the above extraction methods of low molecular weight proteins to circumvent the inherent defects of a single method, while integrating the advantages of other methods, in order to achieve a more ideal extraction effect. SUMMARY

[0008] 1. Technical problems to be solved: How to simply and economically increase the number of low molecular weight (LMW) protein detection in human plasma.

[0009] 2. Technical solutions: In order to solve the above problems, the present application provides a mass spectrometry pretreatment method for low molecular weight proteins in plasma, comprising the following steps: Step 1: Extraction of low molecular weight proteins in plasma: For acetonitrile extraction of plasma, add 100% acetonitrile (AcN) to the plasma to make the final concentration reach 60% and 80% respectively, for trichloroacetic acid (TCA) extraction, add trichloroacetic acid to the plasma to make the final concentration reach 10%, vortex the samples respectively, then rotate at room temperature, then collect the supernatant after centrifugation, dry the supernatant of acetonitrile extraction directly on Speedvac, and dry the supernatant of trichloroacetic acid extraction on Speedvac after desalting on C18 column.

[0010] Step 2: Enzymatic digestion of low molecular weight proteins, reduction alkylation: Add 8M urea to 25mM TEAB, add dried acetonitrile or trichloroacetic acid extract, incubate at room temperature for 2 hours, then dilute to 2M with 25mM TEAB three times the volume, add trypsin, digest at room temperature for 4 hours or more, then reduce the digested sample with a final concentration of 1mM DTT for 30 minutes, incubate at room temperature in the dark, then alkylate with a final concentration of 3mM IAA in the dark for 15 minutes, then quench the reaction again with a final concentration of 1.5mM DTT to obtain a peptide sample.

[0011] Step 3: Desalination of peptides: Load the peptide sample obtained in step 2 into a desalination column, wash with desalination wash solution, then elute with desalination elution solution, collect the eluate containing peptides, and dry to obtain desalted peptides.

[0012] Step 4: Trans liquid chromatography fractionation under basic condition: desalted peptide fragments from step 3 were fractionated by trans liquid chromatography, and each fraction of peptide fragments was collected and dried.

[0013] Step 5: Mass spectrometry detection and protein identification: peptide fragment samples collected from step 4 were injected into liquid chromatography-mass spectrometry instrument for mass spectrometry detection, and mass spectrometry raw data were analyzed using protein database.

[0014] In one embodiment, in step 1, when extracting plasma, dithiothreitol is present, and then 1M dithiothreitol is added to 30mM in plasma during acetonitrile or trichloroacetic acid extraction.

[0015] In one embodiment, in step 2, the extracted sample is dissolved with 300ul 8M urea 25mM TEAB solution, PH≈8.5, and the mass ratio of trypsin to peptide fragments is about 1:200-1:500.

[0016] In one embodiment, in step 3, the mixed peptide fragments after desalting treatment are extracted from low molecular weight proteins extracted from the following samples: 1mg 60% AcN, 1mg 60% AcN_DTT, 1.5mg 80% AcN, 1.5mg 80% AcN_DTT, 1mg 10% TCA and 1mg 10%TCA_DTT.

[0017] In one embodiment, the dithiothreitol is in the form of an aqueous solution, and the dithiothreitol is mixed with water to obtain a dithiothreitol aqueous solution, and the iodoacetamide is in the form of an aqueous solution, and the iodoacetamide is mixed with water to obtain an iodoacetamide aqueous solution.

[0018] In one embodiment, in step 3, the desalting column is a Thermo C18 desalting column, REF:89852, LOT:YL387874, the desalting washing solution is 0.1% formic acid aqueous solution, and the desalting eluent is 40% acetonitrile aqueous solution containing 0.1% formic acid, In one embodiment, the desalting step of the peptide fragments is as follows: Step 31: Acidification: 10% formic acid is added to the mixed peptide fragment solution to be desalted, and the final concentration is adjusted to 0.5%.

[0019] Step 32: Activation: 1500g centrifugation for 1min, the desalting column is treated, 50% DMSO storage solution is centrifuged and discarded, 300ul 100% acetonitrile is added for column activation once, and after passing through the column with 100% acetonitrile, 300ul 60% acetonitrile aqueous solution containing 0.1% formic acid is added.

[0020] Step 33: Equilibration: Equilibrate the column 3 times with 0.1% formic acid in water without acetonitrile.

[0021] Step 34: Injection: Inject the acidified desalting sample into the desalting column and collect the filtrate.

[0022] Step 35: Wash: Repeat the column treatment 3 times with 0.1% formic acid in water without acetonitrile to remove salt impurities from the peptide solution.

[0023] Step 36: Elution: Elute the peptide with 300ul elution salt solution, which contains 0.1% formic acid in 40% acetonitrile in water, and collect the eluate.

[0024] Step 37: Repeat steps 32-36 for the above collected filtrate and combine the eluate with the eluate from step 36.

[0025] In one embodiment, in step 4, the trans liquid chromatography is performed using a liquid chromatograph, and the specific chromatographic conditions are as follows: Column: C18 chromatographic column; Fractionation mobile phase A: 8-12 mM ammonium formate aqueous solution, pH = 9.0; Fractionation mobile phase B: 90% acetonitrile aqueous solution containing 8-12 mM ammonium formate, pH = 9.0.

[0026] In one embodiment, in step 5, the peptide sample collected by step 4 fractionation is resuspended using 5% formic acid aqueous solution, and then injected into a liquid chromatograph-mass spectrometer for mass spectrometry detection, and the specific chromatographic conditions are as follows: Liquid chromatography conditions: Column: Reprosil pure C18 AQ, inner diameter 75 μm, length 25 cm, filled with C18 microspheres, C18 microsphere particle size 1.9 μm; Column temperature: 50°C; Mobile phase A: containing 0.2% formic acid, 3% DMSO, and the rest is deionized water; Mobile phase B: 80% acetonitrile, 0.2% formic acid, 3% DMSO, and the rest is deionized water; Gradient elution: continuously adjusted according to different fractions, total time 90 min, effective gradient 60 min; Injection flow rate 0.3 μL / min.

[0027] Mass spectrometry chromatography conditions: mass spectrometry data is collected using DDA mode, and the parameters are as follows: spray voltage 2.1KV; capillary temperature 320℃; first full scan resolution at 200m / z is 60,000, AGC target: 3e6, Maximum ion time: 50ms, first mass scan range 350-1500; Top N: 20; second full scan resolution at 200m / z is 15,000, AGC target: 1e5, Maximum ion time: 105ms, second scan Fixed first mass 100m / z; fragmentation mode is HCD, fragmentation normalized collision energy is 29, dynamic exclusion time is 30.0s; mass spectrometry raw data analysis is based on a human protein sequence database, and the search parameters are as follows: urea methylation on cysteine is set as a fixed modification parameter; oxidation modification on methionine and acetylation modification on the N-terminus of protein are set as variable modification parameters; specific enzyme cutting selects trypsin, and the maximum number of allowed polypeptide missed cutting is 2.

[0028] The application also provides a mass spectrometry detection pretreatment kit for proteins in plasma, which uses the mass spectrometry detection pretreatment method for low molecular weight proteins in plasma. The first protein precipitation agent is anhydrous acetonitrile. The second protein precipitation agent is trichloroacetic acid. The reducing agent is a 1mol / L dithiothreitol aqueous solution. The alkylation agent is a 0.1mol / L iodoacetamide aqueous solution. The alkylation reaction quenching agent is a 1mol / L dithiothreitol aqueous solution. The desalting column is a C18 desalting column. The desalting cleaning solution is a 0.1% formic acid aqueous solution. The desalting eluent is a 40% acetonitrile aqueous solution containing 0.1% formic acid.

[0029] In the urea solution, the solute is urea, and the solvent is a 25mM, pH≈8.5 TEAB buffer solution. The mass spectrometry detection pretreatment kit described above further comprises anhydrous acetonitrile, a 10% trifluoroacetic acid aqueous solution, fractionation mobile phase A, fractionation mobile phase B, a 5% formic acid aqueous solution, liquid chromatography mobile phase A, liquid chromatography mobile phase B, and a 25mM, pH≈8.5 TEAB buffer solution.

[0030] 3. Beneficial effects: The present application combines 60%, 80% acetonitrile and 10% TCA (with or without DTT) to remove high-abundance proteins in the supernatant of the precipitate in the plasma sample, so as to achieve the purpose of separation, and greatly improve the possibility of discovering clinical disease markers. In the plasma sample, 60%, 80% acetonitrile and 10% TCA (with or without DTT) are added, and the high-abundance proteins such as albumin, globulin, lipoprotein and complement in the plasma are precipitated. The low molecular weight proteins in the supernatant of the plasma precipitated by the above method are combined, and then subjected to chromatographic fractionation by reverse phase liquid chromatography (RPLC) under alkaline pH condition, and finally subjected to mass spectrometry by liquid chromatography mass spectrometry, so as to improve the detection amount of low molecular weight proteins in the plasma sample. The combination of these extracts increases the coverage of the plasma low molecular weight proteome. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1 Figure 1 is a preliminary detection related graph of low molecular weight proteins extracted from plasma by different concentrations of acetonitrile and 10% trichloroacetic acid, wherein Figure 1 Figure 2 is a flow chart of low molecular weight proteins extracted from plasma by different concentrations of acetonitrile precipitation method and 10% TCA precipitation method.

[0032] Figure 1 Figure 3 is a SDS polyacrylamide gel electrophoresis showing low molecular weight proteins in each component extracted from plasma.

[0033] Figure 1 Figure 4 is an LC MS / MS analysis graph of the plasma sample during the extraction process.

[0034] Figure 1 Figure 5 is a graph showing the difference in the number and content of peptides detected by mass spectrometry in each component extracted from plasma.

[0035] Figure 1 Figure 6 is a graph showing the difference in the number and content of peptides detected by mass spectrometry in each component extracted from plasma.

[0036] Figure 2 Figure 7 is a preliminary detection related graph of low molecular weight proteins extracted from plasma by different concentrations of acetonitrile and 10% trichloroacetic acid combined with DTT, wherein Figure 2 Figure 8 is a flow chart of low molecular weight proteins extracted from plasma by different concentrations of acetonitrile precipitation method and 10% TCA precipitation method combined with DTT.

[0037] Figure 2 Figure 9 is a comparison graph of SDS polyacrylamide gel electrophoresis showing low molecular weight proteins in each component extracted from plasma before and after adding DTT.

[0038] Figure 2C is the difference of peptide and content in each component extracted from plasma by mass spectrometry; Fig. 2D is the difference of peptide number before and after adding DTT in each component extracted from plasma by mass spectrometry.

[0039] Figure 3 is 60%, 80% acetonitrile and 10% trichloroacetic acid combined with DTT extraction of low molecular weight protein in plasma ultra deep detection, wherein, Figure 3 A is a flow chart of 60%, 80% acetonitrile and 10% trichloroacetic acid combined with DTT extraction of low molecular weight protein in plasma ultra deep detection; Figure 3 B is the distribution diagram of proteins and peptide numbers identified by mass spectrometry of low molecular weight proteins in each fraction.

[0040] Figure 3 C is the distribution diagram of proteins and peptide numbers identified by mass spectrometry of low molecular weight proteins in plasma extracted by 60%, 80% acetonitrile and 10% trichloroacetic acid combined with DTT in different PSM ranges; Figure 3 D is the distribution diagram of low molecular weight proteins extracted from plasma by 60%, 80% acetonitrile and 10% trichloroacetic acid combined with DTT; Figure 3 E is the analysis of low molecular weight proteins extracted from plasma by 60%, 80% acetonitrile and 10% trichloroacetic acid combined with DTT in clinical application.

[0041] Figure 4 is a schematic diagram of the peptide coverage of the identified low molecular weight proteins.

[0042] Figure 5 is a schematic diagram of the number of peptides and proteins obtained when using acetonitrile and TCA precipitation alone (with or without adding DTT) for single identification. DETAILED DESCRIPTION

[0043] The technical solutions of the present application will be further described in detail below in combination with the drawings and examples.

[0044] Example 1 Preparation of plasma low molecular weight proteins, clinical samples are from a mixture of 100 healthy individual plasma samples, these individuals complete routine health examination, and there is no abnormal result in clinical laboratory tests including infectious screening. All plasma samples were collected within 6 hours after blood collection, 3000g / 10min centrifugation for plasma collection, and stored at-80℃ until use.

[0045] The extraction method of 100ul plasma peptides is as shown in Figure 1 A as follows: Table 1: Extraction method of 100 ul plasma peptides .

[0046] Centrifugal concentration S1-S4 after half an hour of drying, respectively, S1-S4 continue to centrifugal concentration for the next step. About 1-2h salt removal S5 by TCA extraction of samples need to remove TCA in the sample by desalting, the specific steps of desalting as follows: using 50ug desalting column: OMIX gun head on the EP tube, the following each step 3500 rpm instantaneous separation.

[0047] 100% methanol 200ul activation 1 times; Elution salt solution 200ul (60% AcN + 0.1% FA), replacement balance; 200ul 0.1% FA cleaning 2 times, balance; Change tube; Sample, collect the filtrate (filtrate after step one, a total of 3 times); 100ul 0.1% FA cleaning 3-5 times to remove impurities Change tube; 200ul elution salt solution cleaning 1 times; Collect all the elution salt solution drying, for use in the machine; Finally, use 200ul 100% methanol cleaning column for next use Centrifugal concentration again half an hour in advance to open the centrifuge for use, after desalination of TCA extraction sample, namely S5 35-40℃, centrifugal concentration for the next step, about 1-2h.

[0048] The present application provides a kind of mass spectrum detection pre-treatment kit of protein in plasma, comprising: First precipitated protein agent: anhydrous acetonitrile; Second precipitated protein agent: trichloroacetic acid; Enzymatic protease: trypsin; Reducing reagent: 1mol / L dithiothreitol aqueous solution; Alkylation reagent: 0.1mol / L iodine acetamide aqueous solution; Alkylation reaction quenching reagent: 1mol / L dithiothreitol aqueous solution; Desalting column: C18 desalting column; Desalination cleaning fluid: 0.1% formic acid aqueous solution; Desalination eluent: 40% acetonitrile aqueous solution containing 0.1% formic acid.

[0049] The steps of using kit to detect protein concentration are: (1) After centrifugal concentration, each sample was added with 100ul 8M urea solution, and vortexed for 6-8h.

[0050] (2) 0.5% BSA was prepared as a quantitative standard: 10ul 2% BSA (20ug / ul) + 30ul 8M urea solution (3) The 96-well plate was taken out, 10ul of the prepared standard was added, and the second, third, fourth and fifth holes were diluted by 8M urea; (4) 10ul of the prepared protein sample was added in sequence; (5) BCA reagent was prepared in a centrifugal tube: A:B=50:1; (6) 200ul of BCA reagent was added to each well, and it was placed in the dark for 30min to develop color; (7) The absorbance A562 was measured by the enzyme label instrument, and the plate was washed; (8) The standard curve was drawn in Excel table with △A562 as the abscissa and the standard concentration as the ordinate, and the specific parameters are shown in Table 2, Table 2: Parameter table of standard curve .

[0051] The formula of the standard curve was obtained. According to the formula, the concentration of each sample was obtained by substituting the △A562 as the abscissa.

[0052] The BCA quantitative results are as follows: The removal of plasma proteins was observed by SDS-PAGE as shown in Figure 1 B: A 4%-12% precast gel and a balanced plate were installed in the electrophoresis tank. Add ddH2O to check for leaks in the inner tank, and after three minutes of standing, the liquid surface does not change, pour out the new MOPS electrophoresis solution. Carefully and slowly pull out the 15-hole comb, keep the comb hole intact. Use a 10ul pipette to add 1.5ul of protein pre-dyed maeker and each protein sample, each sample has been mixed with 4xLDS to 1xLDS, the original plasma is pre-diluted 50 times with 8M urea, and the original plasma, 60% CAN, 70% CAN, 80% CAN, 90% CAN, and 10% TCA are added in sequence, with a sample volume of 3, 14, 14, 14, 14, and 5ul respectively. When aspirating, take the middle amount in the EP tube and do not aspirate air bubbles, slowly and vertically add the sample to the sample well, first slow and then fast, which is beneficial to the sample to be flat, and it is strictly forbidden to make the sample overflow the sample well because of the too fast sample adding speed. Add recoverable electrophoresis solution to the outer tank. Set the electrophoresis parameters: 150V, 40min.

[0053] Enzymatic digestion and reductive alkylation include the following steps: First, the above plasma separation samples S1-S5 were each added with 300 ul of 25 mM TEAB to dilute urea to 2 M, and the PH test paper was used to test to ensure that the PH of each sample was about 9.

[0054] Second, the above plasma separation samples S1-S5 were each added with Trypsin according to the BCA quantitative results, and the mass ratio of sample: Trypsin was 200-500: 1, i.e., 0.8 ug was added to S1, 0.4 ug was added to S2-S3, and 0.2 ug was added to S4-S5. After vortexing, it was placed at room temperature and shaken overnight for 8-10 h.

[0055] Third, 1M DTT solution was added to the sample to make the working final concentration 1 mM, and after vortexing, it was placed at 25℃ and shaken for 15 min. DTT can open the disulfide bond, and this step must be immediately followed by the next step of IAA (iodoacetamide) operation, otherwise the disulfide bond may be formed again; Fourth, 0.1M IAA was configured in the dark: 10.8 mg of IAA was weighed and mixed with 584 ul of 25 mM TEAB. 0.1M IAA solution was added to the sample to make the final working concentration 3 mM, and after vortexing, it was placed at 25℃ and shaken for 15 min in the dark. IAA blocks sulfhydryl to prevent the disulfide bond from being formed again; Finally, 1M DTT solution was added to make the final working concentration 1.5 mM, and after vortexing, it was placed at 25℃ and shaken for 15 min.

[0056] S1-S5 samples were each selected to be desalted by a desalting column with specifications of 100, 100, 50, 25, and 25 ug. Before desalting, the sample was acidified: the sample was acidified with 10% FA solution to make the final concentration of FA about 0.5%, and the PH was less than 4 The desalting steps of S1-S5 were as follows: First, centrifugal concentration: the centrifuge was turned on half an hour in advance for standby, and the centrifugal concentration was performed at 35-40℃ for about 1-2 h.

[0057] Second, the treatment of the separation sample before LC-MS / MS analysis: 5% FA solution was added to S1-S5 to make the final concentration of the peptide segment of the separation sample about 1-2 ug / ul, and after dissolution, 20000 g centrifugation was performed for 5 min. 40, 30, 20, 8, and 8 ul of supernatant of S1-S5 were added to the chromatographic sample bottle and immediately centrifuged for standby.

[0058] Finally, analysis of the separated sample LC-MS / MS: an appropriate amount of peptide sample was subjected to chromatographic separation by using the Easy nLC1200 chromatographic system at nanoflow rate; the chromatographic column was equilibrated with 100% of A liquid (0.2% FA aqueous solution and 3% DMSO). After the sample was injected into the Trap Column, it was subjected to gradient separation by the chromatographic analysis column. B liquid was a mixed solution of 0.1% FA, 3% DMSO, 80% AcN and water, and the specific parameters used were as follows: liquid phase gradient: 60 min; flow rate: 300 nL / min; column temperature: 50°C. 1 µg of polypeptide was loaded on the chromatographic column made in the laboratory, with a particle size of 1.9 µm; an inner diameter of 75 µm; a length of 25 cm; and a brand of Reprosil pure120ÅC18-AQ.

[0059] Sample separation gradient setting, total gradient 60 min: 0-2 min, 5-10% B; 2-32 min, 10-40% B; 32-35 min, 40%-65% B; 35-37 min, 65%-95% B.

[0060] Mass spectrometry data were collected using the DDA mode of Thermo Q Exactive_HF-X, with the following parameters: spray voltage 2.1 KV; capillary temperature 320°C; primary full scan resolution 60,000 (at 200 m / z), AGCtarget 3e6, Maximum 30 ms; mass scan range 350-1500; secondary full scan resolution 15,000 (at 200 m / z); secondary scan Maximum 50 ms and AGCtarget 1e5; secondary scan Fixed first mass 100 m / z; HCD fragmentation normalized collision energy (NCE) 29; dynamic exclusion time 30.0 s; Top N: the top 20 target ions with the highest ion signals were selected for secondary fragmentation.

[0061] Label-free mass spectrometry analysis was performed by Thermo Q-ExactiveHFX mass spectrometer, and the raw mass spectrometry files were processed by Thermo Proteome Discoverer 2.4 software. The database was searched from Uniprot database for Homo sapiens, and the reviewed (Swiss-Prot) protein sequences were downloaded.

[0062] Other main search parameters were as follows: carbamidomethyl on cysteine was set as a fixed modification parameter; oxidation on methionine, met-loss on N-terminal of protein, and acetyl modification were set as variable modification parameters. Specific enzyme selection was trypsin (Semi), and the maximum number of allowed missed cleavages was 2.

[0063] The total number of protein and peptide identifications of each sample is shown in Table 3, and the ion chromatograms of each sample analysis are shown in 1C. Table 3: Total number of protein and peptide identifications of each sample The specific analysis results of the peptides and proteins are shown in Figure 1 D and 1E.

[0064] During the experiment, we tried to remove high-abundance proteins by adding DTT (dithiothreitol). The results of SDS-PAGE analysis showed that there were large molecular weight proteins in the samples treated by 10% TCA precipitation and 60% AcN precipitation methods, which might interfere with the detection of low molecular weight proteins. In order to further verify the effect of DTT, we compared the changes of samples before and after adding DTT in the SDS-PAGE experiment of 5 kinds of peptidomics extraction methods. The results showed that DTT could effectively remove high-abundance proteins in the supernatant, especially albumin, as shown in Figure 2 B. Therefore, in order to optimize the experimental design and obtain more accurate results, we further compared the differences in mass spectrometry detection results before and after adding DTT in the 5 extraction methods. The specific operation was as follows: First, 1 M DTT solution was added to the plasma sample to make the final concentration reach 30 mM. Then, the mixture was shaken well, and the extraction operation after 15 minutes of action obtained S1-S5_DTT, as shown in Figure 2 A.

[0065] The total number of protein and peptide identifications of the 5 extracted samples after adding DTT is shown in Table 4, Table 4: Total number of protein and peptide identifications of the 5 extracted samples after adding DTT

[0066] Figure 2 C shows the heat map analysis of the 5 methods of extracting peptides after adding DTT, and the difference analysis of mass spectrometry detection results before and after adding DTT is shown in Figure 2 D, The final combination scheme of low molecular weight protein ultra-deep mass spectrometry detection is determined.

[0067] Figure 1 D, and Figure 1 E shows that the differential proteins and differential peptides identified in the 60% AcN, 80% AcN and 10% TCA extracted samples are better than the other two methods. Figure 2 B shows that adding DTT (dithiothreitol) in advance in the plasma can indeed reduce the content of high-abundance proteins to a great extent. Therefore, it is necessary to use the extraction method of adding DTT in the plasma. Therefore, the final extraction method combination of low molecular weight ultra-deep protein detection is: 60% CAN, 60% CAN_DTT, 80% CAN, 80% AcN_DTT, 10% TCA, 10% TCA_DTT, as shown in Figure 3 A.

[0068] The experimental process of ultra-deep mass spectrometry detection is used to detect and analyze the mixed samples of the 5 low molecular weight protein extraction methods.

[0069] This method mainly uses the alkaline pH condition to perform chromatographic fractionation on the peptide sample by reverse phase liquid chromatography (RPLC). The peptides in the sample are gradually eluted according to the size of the hydrophobicity, and are collected in about 60 tubes in turn. Then the peptide sample in each tube is detected by LC-MS / MS. The core technology of this experimental process is to divide the original one-time on-machine detection sample into about 60 samples for detection one by one, so as to improve the detection depth.

[0070] The reagents used in this experiment are shown in Table 5: Table 5: Reagents used Selection of experimental samples: the clinical samples are from the mixture of 100 healthy individual plasma samples, and these individuals complete the routine health examination and have no abnormal results in the clinical laboratory tests including infectious screening. All plasma samples are collected within 6 hours after blood collection, and plasma is collected by centrifugation at 3000g / 10min, and stored at -80℃ until use.

[0071] Sample processing method is as follows: according to the quantitative results of pre-experiment, the following different conditions are taken respectively: 1mg of 60% acetonitrile extraction volume, 1mg of 60% acetonitrile_DTT extraction volume, 1.5mg of 80% acetonitrile extraction volume, 80% acetonitrile_DTT extraction volume, 10% TCA extraction volume and 10% TCA_DTT extraction volume. For sample extraction operation without adding DTT, 60% acetonitrile or 80% acetonitrile is used for extraction. After the sample is mixed with the extraction solvent, it is placed for 15 minutes, then centrifuged at 20,000g for 10 minutes, and the supernatant is collected and dried for standby.

[0072] For samples extracted with 10% TCA (trichloroacetic acid), first extract the sample with 10% TCA, then place the mixture in ice for 30 minutes, and then centrifuge at 20,000g for 10 minutes. After collecting the supernatant, remove the salt by appropriate method, and dry for treatment. For sample extraction with DTT (dithiothreitol) added, 1M DTT solution is added to the plasma sample to make the final concentration 30mM. After mixing well for 15 minutes, the subsequent treatment is completed according to the same operation steps (i.e. centrifugation, supernatant collection, drying) as described above.

[0073] Dissolution of sample: 300ul of 8M urea in 25mM TEAB is added to each of the 5 dried samples for 2h.

[0074] Proteolysis, reduction alkylation: then dilute the urea to 2M with 900μL of 25mM TEAB (pH 8.5), and add 5μg of trypsin to each sample, continue to rotate at room temperature for 12-24 hours. The next day, add 1M DTT aqueous solution to each sample to make the final concentration of DTT 1mM, react at room temperature for 1 hour, then add 0.1M IAA aqueous solution to make the final concentration of IAA 3mM, react at room temperature for half an hour, then add 1M DTT aqueous solution to make the final concentration of DTT 2mM, react at room temperature for 15 minutes, and then quench the remaining IAA to obtain it.

[0075] Desalination of sample peptides includes the following steps: ①Add 1 / 19 volume of 10% FA aqueous solution to the 5 peptide samples obtained in the above "proteolysis" to make the final volume concentration of TFA (trifluoroacetic acid) 0.5%.

[0076] ②Take Thermo desalting column (Thermo C18 desalting column, REF: 89852).

[0077] ③ After passing the column with 300ul 100% acetonitrile, add 300ul 60% acetonitrile solution containing 0.1% formic acid, and then pass the column with 300ul 0.1% formic acid solution without acetonitrile to balance the column.

[0078] ④ Take the peptide segment sample in each sample and add it to the column and pass the column.

[0079]

[0080] ⑥ Add 300ul 40% acetonitrile solution containing 0.1% formic acid to the column and collect the eluted peptide segments.

[0081] ⑦ Combine all desalted eluted peptide segments from the 6 samples and perform vacuum drying to obtain desalted mixed peptide segments.

[0082] A liquid chromatography system (Shimadzu) and a C18 chromatography column (XBridge C18 column 3.5um, 4.6mm x 250mm, Waters) were used. In the liquid phase system, the fractionation mobile phase A: 10mM ammonium formate aqueous solution (NH4COOH), pH 8.0; fractionation mobile phase B: 10mM ammonium formate containing 90% acetonitrile aqueous solution, pH 8.0.

[0083] The desalted mixed peptide segments were subjected to RPLC fractionation under alkaline pH conditions, including the following steps: ① Each sample of 4mg desalted peptide segments based on protein was dissolved and resuspended with 0.5mL mobile phase A.

[0084] ② Prepare the liquid chromatography system and clean the system with at least 50mL fractionation mobile phase B, and then balance the system with at least 50mL fractionation mobile phase A. ③ Load the 0.5mL mobile phase A to resuspend the 4mg desalted peptide segments based on protein.

[0085] ④ Use gradient mobile phase to fractionate the sample, and the specific gradient design is as follows: 0.01-10 minutes, 5%-5% mobile phase B; 10-40 minutes, 5-25% mobile phase B; 40-45 minutes, 25-50% mobile phase B; 45-50 minutes, 50-65% mobile phase B; 50-55 minutes, 65-95% mobile phase B; 55-60 minutes, 95-95% mobile phase B.

[0086] ​Flow rate: 1 mL / min.

[0087] 5. The collected fraction tubes were dried.

[0088] The peptides in each fraction tube after drying were detected by mass spectrometry and protein identification: The collected fraction samples were resuspended with about 1 μg of sample in 5% formic acid solution, and then loaded onto a mass spectrometer (Thermo Q Exactive HF X) for detection. The mass spectrometer was connected in series with a nanoliter liquid chromatography system (Thermo EASY nLC 1200).

[0089] Liquid chromatography conditions: Laboratory-made chromatographic column: Reprosil pure C18 AQ, inner diameter 75 μm, length 25 cm, filled with C18 microspheres, C18 microsphere particle size 1.9 μm; column temperature: 50°C.

[0090] Mobile phase A: contains 0.2% formic acid, 3% DMSO, the rest is deionized water.

[0091] Mobile phase B: 80% acetonitrile, 0.2% formic acid, 3% DMSO, the rest is deionized water; gradient elution: continuously adjusted according to different fractions. Elution time is 60 minutes; injection flow rate is 0.3 μL / min.

[0092] Mass spectrometry conditions: Mass spectrometry data was collected using the DDA mode of Thermo Q Exactive_HFX, with the following parameters: spray voltage 2.1 KV; capillary temperature 320°C; primary full scan resolution 60,000 (at 200 m / z), AGC target: 3e6, Maximum ion time: 50 ms, primary mass scan range 350 1500; Top N: 20; secondary full scan resolution 15,000 (at 200 m / z), AGC target: 1e5, Maximum ion time: 105 ms, secondary scan Fixed first mass 100 m / z; fragmentation mode HCD, fragmentation normalized collision energy (NCE) 29, dynamic exclusion time 30.0 s.

[0093] Search of peptides: Mass spectrometry raw data analysis was processed by Thermo Proteome Discoverer 2.5 software with database searching. Reviewed human protein sequences in Swiss Prot protein database were used as database. Main search parameters were as follows: carbamidomethyl on cysteine was set as fixed modification parameter; oxidation modification on methionine, acetyl on N-terminal of protein were set as variable modification parameters. Trypsin was selected as specific enzyme cleavage, and the maximum number of allowed missed cleavages was 2.

[0094] Results: Equal amount of peptides from the five extraction samples were combined and fractionated after digestion for in-depth proteomic analysis to obtain the identification results, the amount of proteins identified in each fraction, as shown in Figure 3 B. That is, by plasma acetonitrile combined with TCA (with or without DTT) precipitation method, the number of identified low molecular weight peptides in plasma can be increased from 3154 to 5890, as shown in Figure 3 A and Figure 5 5890 peptides were identified from 820 proteins, as shown in Figure 3 A. Among them, 2343 peptides were scanned only once (PSM=1), 1131 peptides were detected twice (PSM=2), and the rest of the peptides were detected more than twice.

[0095] At the protein level, 214 proteins were “PSM=1”, 369 proteins were “PSM=2”, and the rest of the proteins had higher PSM values, as shown in Figure 3 C. If the proteins in the extract are full-length, as shown in the SDS-PAGE results of the 60% acetonitrile extract, as shown in Figure 1 B, the coverage of the identified peptides is often higher; while if the identified peptides only come from fragments of proteins in the extract, the coverage of the protein is relatively low. The data shows that about 50% of the identified proteins have a coverage of less than 10%, and about 25% have a coverage between 10% and 30%, as shown in Figure 4 , which indicates that most protein species come from fragments.

[0096] To evaluate the depth of the analysis, we used two data sets of plasma proteins with relative abundance (PSM) or absolute concentration as reference, and found that the actual level of some proteins in plasma is very low, which indicates that this in-depth proteomic analysis is indeed very deep, as shown in Figure 3 D. Gene ontology analysis shows that the proteins identified from the extract are enriched in a subset of certain cellular components, molecular functions and biological processes, as shown in Figure 3E. To explore the factors that might affect the presence of these low-molecular-weight protein species, we first compared their abundance to the levels of their intact proteins in whole plasma or in public databases, both of which showed significant correlation, indicating that the levels of low-molecular-weight protein species are related to their total levels or the levels of their precursor proteins. We then investigated whether longer proteins are more likely to produce low-molecular-weight protein species, however, there was no significant correlation between the number of amino acids of PSMs and precursor proteins.

[0097] The present application provides a method for processing mass spectrometry of low-molecular-weight proteins in plasma. There are many ways to achieve the technical solutions of the method and approach. The above description is only the preferred embodiment of the present application. It should be noted that for ordinary skilled in the art, without departing from the principles of the present application, several improvements and refinements can be made, which should be considered as the protection scope of the present application. The components not explicitly described in the embodiments can be implemented using existing technology.

Claims

1. A method for pre-treatment of low molecular weight proteins in plasma by mass spectrometry detection, comprising the following steps: Step 1: Extraction of low molecular weight proteins from plasma: For acetonitrile extraction of plasma, 100% acetonitrile was added to the plasma to a final concentration of 60% and 80%, respectively. For trichloroacetic acid extraction, trichloroacetic acid was added to the plasma to a final concentration of 10%. These samples were vortexed and then rotated at room temperature. After that, the samples were centrifuged and the supernatant was collected. The supernatant of acetonitrile extraction was directly dried on a Speedvac, while the supernatant of trichloroacetic acid extraction was first desalted by passing through a C18 column and then dried on a Speedvac. Step 2: Enzymatic digestion and reductive alkylation of low molecular weight proteins: 8M urea was added to 25mM TEAB, and dried acetonitrile or trichloroacetic acid extract was added. The mixture was incubated at room temperature for 2 hours, and then diluted to 2M with three times the volume of 25mM TEAB. Trypsin was added and digested at room temperature for more than 4 hours. After that, the digested sample was reduced with a final concentration of 1mM DTT for 30 minutes, incubated at room temperature in the dark, and then alkylated with a final concentration of 3mM iodoacetamide in the dark for 15 minutes. After completion, the reaction was quenched again with a final concentration of 1.5mM DTT to obtain peptide samples. Step 3: Peptide desalting: Load the peptide sample obtained in step 2 onto the desalting column and use desalting wash solution for desalting Washing, followed by adding desalting eluent for elution, collecting the eluent containing the peptides, and drying to obtain the desalted peptides; Step 4: Reverse liquid phase chromatography fractionation under alkaline conditions: The desalted peptides obtained in step 3 are fractionated by reverse liquid phase chromatography, and the peptide fractions obtained are collected and dried; Step 5: Mass spectrometry detection and protein identification: The peptide samples collected by fractionation in step 4 are injected into a liquid chromatography-mass spectrometer for mass spectrometry detection, and the raw mass spectrometry data are analyzed using a protein database.

2. The method for pre-treatment of low molecular weight proteins in plasma by mass spectrometry detection according to claim 1, wherein: In step 1, if dithiothreitol is present during plasma extraction, 1 M dithiothreitol is first added to the plasma to a concentration of 30 mM during the acetonitrile or trichloroacetic acid extraction.

3. The method for pre-treatment of low molecular weight proteins in plasma by mass spectrometry detection according to claim 1, wherein: In step 2, the extracted sample was dissolved in 300 μl of 8 M urea 25 mM TEAB solution, pH ≈ 8.5, and the mass ratio of trypsin to peptide was approximately 1:200-1:

500.

4. The method for pre-treatment of low molecular weight proteins in plasma by mass spectrometry detection according to claim 1, wherein: In step 3, the desalted peptide mixture was extracted from the following samples: 1 mg 60% AcN, 1 mg 60% AcN_DTT, 1.5 mg 80% AcN, 1.5 mg 80% AcN_DTT, 1 mg 10% TCA, and 1 mg 10% TCA_DTT extracted low molecular weight proteins.

5. The method for pre-treatment of low molecular weight proteins in plasma by mass spectrometry detection according to claim 2, wherein: The dithiothreitol exists in the form of an aqueous solution, and the dithiothreitol is mixed with water to obtain a dithiothreitol aqueous solution. The iodoacetamide exists in the form of an aqueous solution, and the iodoacetamide is mixed with water to obtain an iodoacetamide aqueous solution.

6. The method for pre-treatment of low molecular weight proteins in plasma by mass spectrometry detection according to claim 1, wherein: In step 3, the desalting column is a Thermo Fisher C18 desalting column, REF: 89852, LOT: YL387874, the desalting cleaning solution is a 0.1% formic acid aqueous solution; the desalting eluent is a 40% acetonitrile aqueous solution containing 0.1% formic acid, The method for pre-treatment of low molecular weight proteins in plasma by mass spectrometry detection according to claim 6, wherein the desalting step of the peptide segment is as follows: Step 31: Acidification: Add 10% formic acid to the desalted peptide mixture to adjust the final concentration to 0.5%; Step 32: Activation: Treat the desalting column by centrifugation at 1500 g for 1 min. Discard the 50% DMSO preservative and add 300 μl of 100% acetonitrile to activate the column once. After passing the column through 100% acetonitrile, add 300 μl of 60% acetonitrile solution containing 0.1% formic acid. Step 33: Equilibration: Equilibrate the column three times with 0.1% formic acid in water without acetonitrile. Step 34: Adding samples: Add the acidified sample to be desalted to the desalting column in batches and collect the filtrate; Step 35: Cleaning: Repeat the column treatment three times with 0.1% formic acid aqueous solution without acetonitrile to remove salt impurities in the peptide solution; Step 36: Elution: Add 300 μl of elution salt solution (40% acetonitrile aqueous solution containing 0.1% formic acid) to elute the peptide fragments and collect the eluate; Step 37: Repeat steps 32 to 36 with the filtrate collected above to collect the eluate, and combine it with the eluate in step 36.

7. The method for pre-treatment of low molecular weight proteins in plasma by mass spectrometry detection according to claim 1, wherein: In step 4, the reverse liquid chromatography is performed using a liquid chromatograph, and the specific chromatographic conditions of the liquid chromatograph are as follows: chromatographic column: C18 chromatographic column; fractionation mobile phase A: 8-12 mM ammonium formate aqueous solution, pH = 9.0; fractionation mobile phase B: 90% acetonitrile aqueous solution containing 8-12 mM ammonium formate, pH = 9.

0.

8. The method for pre-treatment of low molecular weight proteins in plasma by mass spectrometry detection according to claim 8, characterized in that: In step 5, the peptide sample collected by fractionation in step 4 is resuspended in 5% formic acid aqueous solution and injected into a liquid chromatography-mass spectrometer for mass spectrometry detection. The specific chromatographic conditions are as follows: Liquid chromatography conditions: Column: Reprosil pure C18 AQ, inner diameter 75 μm, length 25 cm, packed with C18 microspheres, particle size of C18 microspheres 1.9 μm; Column temperature: 50°C; mobile phase A: 0.2% formic acid, 3% DMSO, balance deionized water; mobile phase B: 80% acetonitrile, 0.2% formic acid, 3% DMSO, balance deionized water; gradient elution: adjusted continuously according to different fractions, total time 90 min, effective gradient 60 min; injection flow rate 0.3 μL / min; Mass spectrometry data were collected in DDA mode with the following parameters: spray voltage 2.1 kV; capillary temperature 320°C; primary full scan resolution 60,000 at 200 m / z, AGC target 3e6, maximum ion time 50 ms, primary mass scan range 350–1500; Top N 20; secondary full scan resolution 15,000 at 200 m / z, AGC target 1e5, maximum ion time 105 ms, secondary scan fixed first mass 100 m / z; HCD fragmentation mode, fragmentation normalized collision energy 29, dynamic exclusion time 30 .0s; the mass spectrometry raw data analysis was based on human protein sequences as the database, and the search parameters were as follows: carbamidomethylation on cysteine ​​was set as a fixed modification parameter; oxidation modification on methionine and acetylation modification on the protein N-terminus were set as variable modification parameters; trypsin was selected as the specific enzyme cleavage, and the maximum number of missed cleavages allowed for the peptide was 2.

9. A kit for pretreatment of plasma proteins by mass spectrometry, comprising the method for pretreatment of plasma low molecular weight proteins by mass spectrometry according to any one of claims 1 to 9, characterized in that: include: First protein precipitation agent: anhydrous acetonitrile; Second protein precipitation agent: trichloroacetic acid; enzymatic protease: trypsin; Reducing agent: 1 mol / L dithiothreitol aqueous solution; alkylating agent: 0.1 mol / L iodoacetamide aqueous solution; alkylation reaction quenching reagent: 1 mol / L dithiothreitol aqueous solution; desalting column: C18 desalting column; desalting cleaning solution: 0.1% formic acid aqueous solution; desalting eluent: 40% acetonitrile aqueous solution containing 0.1% formic acid.

10. In the urea solution, the solute is urea, and the solvent is 25 mM TEAB buffer with a pH of ≈8.

5. The mass spectrometry pretreatment kit further includes anhydrous acetonitrile, 10% trifluoroacetic acid aqueous solution, fractionation mobile phase A, fractionation mobile phase B, 5% formic acid aqueous solution, liquid mobile phase A, liquid mobile phase B, and 25 mM TEAB buffer with a pH of ≈8.5.

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