Plasma proteomics pretreatment method

Abstract

The invention relates to a plasma proteomics pretreatment method, and relates to the field of proteomics detection. According to the technical scheme, the removal efficiency of serum albumin reaches 50% or above, meanwhile, the influence on detection of other proteins is small, and the performance is equivalent to or even better than that of a removal kit.

Description

Technical Field

[0001] The invention relates to a plasma proteomics pre-treatment method, belonging to the technical field of proteomics. Background Art

[0002] UHPLC-MS is a commonly used technical method in proteomics. In the process of plasma proteomics research, sample pretreatment is a crucial step, which can significantly affect the quality and results of subsequent analysis. However, due to the interference of high-abundance plasma proteins (especially serum albumin (ALB) accounting for more than 80%), the detection of low-abundance proteins with more biological significance is limited. Low-abundance protein enrichment or high-abundance protein removal methods are usually used to improve the depth of plasma proteomics identification. The former mainly achieves targeted enrichment of a small amount of protein through specific antibody capture or nanoparticle capture, which requires specific antibodies, has a limited enrichment quantity and is relatively expensive. The latter includes dye ligands, protein ligands or immune antibody capture removal and peptide ligand library capture removal. Although there are mature kits, there is non-specific removal and the removal cost is relatively high. Organic solvent precipitation is a relatively simple protein precipitation method with high resolution and no need for desalting. It is also used in proteomics, but due to its poor selectivity for proteins, it is less used in plasma proteomics. Summary of the invention

[0003] The technical problem to be solved by the present invention is that during the pretreatment of proteomics samples, high-abundance proteins account for a large proportion and affect the results of the detection of low-abundance proteins. The separation selectivity in the process of precipitation with organic solvents is poor, making it difficult to obtain low-abundance proteins with more biological significance. The pretreatment method in the technical solution of the present invention performs pretreatment of plasma proteins, and by adjusting the proportion of organic reagent precipitation and pH, the removal efficiency of serum albumin reaches more than 50%, while having little effect on the detection of other proteins, and the performance is equivalent to or even better than that of the removal kit.

[0004] A plasma proteomics pre-processing method comprises the following steps:

[0005] Step 1, after diluting the plasma, add a pH adjuster to adjust the pH to a range of 4.0-6.0;

[0006] Step 2, mixing the organic reagent with the sample obtained in step 1 in a volume ratio of 4:5-1:3, and after being evenly dispersed, centrifuging to remove the supernatant;

[0007] Step 3, the precipitate obtained in step 2 is redissolved, then subjected to reduction treatment, alkylation treatment, and enzymatic hydrolysis, and then dried to obtain a polypeptide for proteomics mass spectrometry detection.

[0008] In the step 1, the pH regulator is acetic acid, and the dilution process uses an NH4HCO3 solution with a pH value of 8.0-9.0.

[0009] In the step 2, the centrifugation speed is 4000-6000 rpm and the centrifugation time is 10-30 min.

[0010] In the step 2, the organic reagent is methanol, acetonitrile or a mixture of the two.

[0011] In step 3, the re-dissolution process uses a 4-8 M urea aqueous solution.

[0012] In the reduction and alkylation process, the reducing agent used was TCEP and the alkylating agent was IAA.

[0013] The pretreatment method is used to remove serum albumin and / or obtain low-abundance proteins.

[0014] The low-abundance protein is selected from one or more of C2, CFD, and MASP2 complement-related proteins, or is selected from one or more of F11, CPB2, SERPINA5, VWF, IGFBP3, and CD14 proteins. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 :Flowchart of plasma proteomics pre-processing

[0016] Figure 2 :Total ion current data quality control results

[0017] Figure 3 :Statistical results of the number of plasma proteins identified and the proportion of serum albumin

[0018] Figure 4 Quantitative results of different gradient proteins

[0019] Figure 5 :Protein band distribution results

[0020] Figure 6 :Protein band distribution results

[0021] Figure 7 :Identification and distribution of plasma proteins and the proportion of serum albumin and immunoglobulin

[0022] Figure 8 : Plasma gradient protein quantitative results DETAILED DESCRIPTION

[0023] Five fresh frozen plasma samples from healthy subjects (freeze-thaw times less than 3 times) were used to prepare mixed plasma at a ratio of 1:1. After dilution 10 times, the effects of the plasma proteomics protein identification performance under precipitation conditions of no removal group (KA), different ratios of organic reagent to sample (4:5 (MA), 1:1 (MB), 1:3 (MC) and pH 8.5 (MD) were compared.

[0024] More specifically:

[0025] 10ul of diluted mixed plasma was taken, diluted 10 times with NH4HCO3 solution of pH8.5, and a small amount of acetic acid (MA-MC) was added to adjust the pH of the solution to 5.0 (acetic acid was not added to adjust the pH in the MD group). Except for KA, a certain amount of pre-cooled organic reagent (methanol) was added to the sample in proportion in other groups. The volume ratio of organic reagent to sample was 4:5 (MA), 1:2 (MB), 1:3 (MC) and 1:2 (MD). The samples were shaken and mixed, ultrasonicated for 10 minutes, centrifuged at 4℃, 5000rpm for 20 minutes, and the supernatant was discarded. The precipitate was re-dissolved with 30ul6-8M urea, and after BCA quantification, 50ug of protein was taken from each group into a new EP tube, and a certain amount of TCEP was added. After IAA reduction alkylation treatment for 30min-1h, Trypsin and Lys-C were added at a ratio of 1:100 and enzymatic hydrolysis was carried out at 37℃ overnight. After adding TFA to terminate the reaction, the salt was removed, and the obtained peptide was vacuum dried and concentrated and stored at -80 for loading. The detection conditions for the main protein product of the processed sample for HPLC-MS determination are: the peptide sample dry powder was re-dissolved with 20ul 0.1% formic acid water, and the mass spectrometry detection was performed using DDA (data-dependent scanning mode) mode. The specific conditions are as follows:

[0026] Instrument model: EASY-nLC 1200+Q Exactive TM HF Instruments

[0027] Column specifications: C18 column (20cm / 75um, 1.9um beads)

[0028] Peptide entry amount: ≈500ng (peptide)

[0029] Liquid chromatography gradient elution conditions: Solution A: 0.5% formic acid; Solution B: 80% acetonitrile

[0030] The elution procedure is:

[0031]

[0032] Mass spectrometry parameters:

[0033] Scanning mass-to-charge ratio range: 350-1500m / z (primary) 100m / z (secondary)

[0034] Scan type: MS1-ddMS (Top10)

[0035] Detector resolution: 60000 (primary) 30000 (secondary)

[0036] HCD collision energy: 32%

[0037] AGC (automatic gain value): 1E5

[0038] Dynamic exclusion time: 30s

[0039] Mass spectrometry scanning mode: DDA (data dependent scanning)

[0040] Offline data retrieval method

[0041] Database: Homosapiens(Human)

[9606] , proteome:up000005640

[0042] Search software: Maxquant 2.0

[0043] Search parameters:

[0044] Modification:Oxidation(M),Acetyl(protein N-terminal),Carbamidomethyl(C);

[0045] Digestion:Trypsin / P

[0046] Max.missed:2

[0047] Min.peptide length:6

[0048] Label-free quantification:LFQFDR:0.01

[0049] The quality control conditions of the test results are: total ion current base peak response (TIC) greater than 1E9, base peak retention time shift less than or equal to 1min, and three repeated correlations on the machine ≥ 0.95.

[0050] The proteomics test data showed that the total ion current base peak of each group was above 1E09, and the quality control was qualified. The total ion current signal response and secondary signal response of the non-removal group (KA) were higher than those of the other removal groups, which may be related to the non-removal of high-abundance proteins. Although the primary signal of the pH8.5 precipitation group was equivalent to that of the non-removal group, the secondary signal was lower than that of the non-removal group ( Figure 2 ).

[0051] Protein identification results ( Figure 3 ) showed that compared with the non-removal group (KA), the number of organic reagent histone identifications increased, and the proportion of serum albumin decreased from 70% to less than 20%. The ability of serum albumin precipitation at pH 5.0 (MB) and pH 8.5 (MD) was equivalent. When the ratio of organic reagent to sample was 4:5, the serum albumin removal efficiency was slightly poor. The higher the ratio of organic reagent, the worse the removal effect. The maximum removal could reach 15%. There was no significant difference in the number of histone identifications after removal ( Figure 3 ), removing some serum albumin can increase the amount of plasma protein detected. Although the total number of plasma protein identifications in the MD (pH = 8.5) precipitation group was not much different, the detection of F11, PROS1, APOC3, HP, and ALB protein markers did not increase, which may simultaneously remove some target proteins, making it unfavorable for detection. Different pH conditions are selective for proteins with different isoelectric points, and some low-abundance proteins may be removed under pH 8.5 conditions, affecting the detection performance.

[0052] Quantitative results of the same protein between different methods ( Figure 4 ) showed that the quantitative distribution of proteins among the groups was not much different, and the detection of low-abundance proteins increased after organic precipitation removed part of the serum albumin. Except for ALB, the protein quantification of the MB group (pH = 5.0, organic reagent to sample ratio = 1:2) was close to that of the non-removal group, with less influence on quantification and the best stability within the group.

[0053] And the plasma protein distribution results showed ( Figure 5 ) Compared with the non-removal group (KA), the MA-MD groups all had different degrees of reduced serum albumin (ALB) expression levels. The MA (pH5.0, 5:4) group had the lowest removal efficiency, followed by the MD (pH8.5, 3:1) group. The nonspecific removal was more obvious in the MC and MD groups. In addition to serum albumin, other unknown protein bands were removed. Therefore, pH5.0 and the ratio of organic reagent to sample of 1:2 had the best effect, which can be used for plasma proteomics pretreatment.

[0054] Performance comparison of similar methodologies

[0055] In order to verify the reliability of the method, two commonly used ALB dye removal kits and ALB and IG six protein removal kits were selected for comparison. At the same time, in order to reduce the non-specific loss of proteins, the precipitation condition of pH 8.5 was added to the above pH 5.0 conditions. The proteins removed by the two were combined as the optimization conditions and the optimal performance conditions were selected as the plasma proteomics pre-processing conditions. Protein band distribution results ( Figure 6) showed that compared with the ALB single removal kit, the protein richness obtained by the method of the present invention is higher, the ALB kit has poor ALB removal efficiency and obvious nonspecific removal, and the plasma protein loss rate is high. The results of plasma protein detection and high-abundance protein proportion showed that the number of plasma proteins identified by the method of the present invention is more than that of the kit, but the proportion of serum albumin detected is slightly higher than that of the kit group, and the proportion of immunoglobulins is lower than that of the ALB single removal group but significantly higher than that of the ALB, IG six-protein kit group. Under the current mass spectrometry detection conditions, the plasma protein identification of the method of the present invention is slightly better than that of the kit. The kit method may have nonspecific adsorption and large protein loss.

[0056] Plasma protein quantitative results ( Figure 8 ) showed that compared with the ALB, IG six-protein removal kit, the total amount of plasma proteins identified by the method of the present invention was significantly improved, and the number of some low-abundance proteins F11, PROS1, and APOC3 in different gradients remained basically unchanged. The detected proteins were highly consistent and the low-abundance F11 protein could be detected in all three methods, and the abundance was not much different. The combined optimization of the two precipitation conditions did not improve the performance but increased the complexity of the process. The processing method of the present invention is better.

[0057] Comprehensive data results show that the present invention achieves a removal efficiency of more than 50% for serum albumin by adjusting the ratio of organic reagent precipitation and pH, while having little effect on the detection of other proteins, and the performance is comparable to or even better than that of the removal kit.

Claims

1. A plasma proteomics pre-processing method, characterized in that: The steps include: Step 1, after diluting the plasma, add a pH adjuster to adjust the pH to a range of 4.0-6.0; Step 2, mixing the organic reagent with the sample obtained in step 1 in a volume ratio of 4:5-1:3, and after being evenly dispersed, centrifuging to remove the supernatant; Step 3, the precipitate obtained in step 2 is redissolved, then subjected to reduction treatment, alkylation treatment, and enzymatic hydrolysis, and then dried to obtain a polypeptide for proteomics mass spectrometry detection.

2. The plasma proteomics pre-processing method according to claim 1, characterized in that: In the step 1, the pH regulator is acetic acid, and the dilution process uses an NH4HCO3 solution with a pH value of 8.0-9.

0.

3. The plasma proteomics pre-processing method according to claim 1, characterized in that: In the step 2, the centrifugation speed is 4000-6000 rpm and the centrifugation time is 10-30 min.

4. The plasma proteomics pre-processing method according to claim 1, characterized in that: In the step 2, the organic reagent is methanol, acetonitrile or a mixture of the two.

5. The plasma proteomics pre-processing method according to claim 1, characterized in that: In step 3, the re-dissolution process uses a 4-8 M urea aqueous solution.

6. The plasma proteomics pre-processing method according to claim 1, characterized in that: In the reduction and alkylation process, the reducing agent used was TCEP and the alkylating agent was IAA.

7. The plasma proteomics pre-processing method according to claim 1, characterized in that: The pretreatment method is used to remove serum albumin and / or obtain low-abundance plasma proteins.

8. The plasma proteomics pre-processing method according to claim 1, characterized in that: The low-abundance plasma protein is selected from one or more of C2, CFD, and MASP2 complement-related proteins, or is selected from one or more of F11, CPB2, SERPINA5, VWF, IGFBP3, and CD14 proteins.

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