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CN-121994952-A - Sample pretreatment method for low molecular weight protein in plasma

CN121994952ACN 121994952 ACN121994952 ACN 121994952ACN-121994952-A

Abstract

The invention discloses a sample pretreatment method of low molecular weight protein in plasma, which comprises the steps of diluting the plasma with ammonium formate solution, loading the diluted plasma to a C18 solid phase extraction column, and sequentially and gradient eluting and splitting a complex sample by adopting seven ACN/AmF solutions with different proportions according to the sequence from low to high, wherein the volume ratio of ACN is 15-90%, and finally realizing high-efficiency enrichment and accurate identification of the low molecular weight protein in the plasma by combining with subsequent reductive alkylation, enzymolysis and mass spectrometry. The method not only balances the experimental workload and the identification effect, obviously improves the identification quantity and coverage of the low molecular weight protein in the blood plasma, but also makes up the defects of the prior art, provides a more practical pretreatment means for the study of the proteomics of the blood plasma, and lays an important foundation for the excavation of the related biomarkers of the diseases, the assisted clinical diagnosis and the medical study because the low molecular weight protein is a key biomarker resource for the diagnosis of the diseases.

Inventors

  • LI QINGRUN
  • QIAN SHANSHAN

Assignees

  • 中国科学院上海高等研究院

Dates

Publication Date
20260508
Application Date
20260123

Claims (10)

  1. 1. A method for sample pretreatment of low molecular weight proteins in plasma, comprising the steps of: S1, enriching low molecular weight proteins in plasma, namely preparing a C18 solid phase extraction column, washing the column with methanol, balancing the column with an ammonium formate solution, diluting the plasma with 5-20 mM ammonium formate solution, loading the diluted plasma to the C18 solid phase extraction column, washing the column with the ammonium formate solution, sequentially and gradient eluting with seven ACN/AmF solutions with different proportions according to the sequence of the ACN volume ratio from low to high, wherein the ACN volume ratio is 15% -90%, and collecting and drying eluent; S2, enzymolysis of enriched protein, namely re-dissolving the dried eluent with 8M urea/100 mM Tris buffer solution, sequentially carrying out TCEP reduction and IAA alkylation treatment, diluting urea concentration, adding trypsin for enzymolysis, and stopping reaction with formic acid to obtain a peptide sample; S3, desalting the peptide fragment, namely loading the peptide fragment sample to a C18 desalting column, washing by 0.2% TFA/H 2 O, and eluting by 90% ACN/0.2% TFA; S4, drying the eluent in the step S3, re-dissolving the eluent by using 0.1% FA/H 2 O, obtaining mass spectrum data through LC-MS/MS analysis, and utilizing Spectronaut software to search a database for analysis.
  2. 2. The sample pretreatment method according to claim 1, wherein the C18 solid phase extraction column in the step S1 is prepared by a laboratory self-control method, and the preparation process comprises the steps of taking a C18 desalting column, adding a mixed solution of 10mg of C18 packing and 100 mu L of methanol, and centrifuging for 2min at a rotating speed of 500rcf to obtain the C18 solid phase extraction column with a column bed height of 15 mm.
  3. 3. The method for pretreatment of a sample according to claim 1, wherein the concentration of the ammonium formate solution in the step S1 is 10mM, the pH value is 2 to 4, and the washing volumes of the methanol and the ammonium formate solution are 100 to 200. Mu.L.
  4. 4. The method according to claim 1, wherein the dilution ratio of plasma to ammonium formate solution in step S1 is 1 (8-12).
  5. 5. The method according to claim 1, wherein the ACN/AmF solution of different proportions in step S1 is based on an ammonium formate solution of 10mM and pH 3.0, and the ACN is 15%, 30%, 35%, 37.5%, 40%, 50%, 90% ACN/AmF solution in sequence, and each elution solution has a volume of 100 to 200 μl.
  6. 6. The method of claim 1, wherein the eluent in step S1 is dried under vacuum of 0.01mbar at 25℃for 2h in Speedvac.
  7. 7. The method of claim 1, wherein the pH of the 8M urea/100 mM Tris buffer solution in step S2 is 8.5, the final concentration of TCEP is 5mM, the reduction reaction condition is 30min at room temperature, the final concentration of IAA is 10mM, and the alkylation reaction condition is 20min in the dark.
  8. 8. The method for pretreatment of a sample according to claim 1, wherein in step S2, the protein mixture is diluted four times with 100mM Tris buffer, the mass ratio of trypsin to protein is 1:25 to 1:200, the enzymolysis is carried out overnight, and the final concentration after formic acid termination reaction is 5%.
  9. 9. The method according to claim 1, wherein the centrifugation speed of the C18 desalting column in the step S3 is controlled at 500rcf, and the sample is loaded after 200. Mu.L of methanol activation and 200. Mu.L of 0.2% TFA/H 2 O equilibration, and the washing process is performed twice with 200. Mu.L of 0.2% TFA/H 2 O.
  10. 10. The method according to claim 1, wherein the LC-MS/MS analysis in the step S4 comprises the steps of separating by using an Easy nLC 1200 chromatographic column, wherein the chromatographic column is ReproSil-Pur 120C 18-AQ chromatographic column, the mobile phase A is 0.1% FA/H 2 O, the mobile phase B is 0.1% FA/80% ACN, the flow rate is 300nL/min, the column temperature is 55 ℃, the gradient time is 60min, the mass spectrum data are collected by using a DIA mode, the spray voltage is 2.1kV, the capillary temperature is 320 ℃, the primary full scan resolution is 120000, the maximum injection time is 50MS, the mass scan range is 350-1500, the secondary scan resolution is 15000, the HCD collision energy is 32%, the mass scan range is 200-2000, the maximum injection time is 54MS, the parameters of a Spctronaut software search library are Carbamidomethyl modification on cysteine is fixed modification, oxidation on methionine is variable modification, the specific enzyme is Trypsin, and the allowable leakage number is 2.

Description

Sample pretreatment method for low molecular weight protein in plasma Technical Field The invention relates to the field of enrichment analysis of low molecular weight proteins in plasma, in particular to a sample pretreatment method of the low molecular weight proteins in the plasma. Background Low molecular weight proteins are important components in plasma and have been shown to be closely related to a variety of human diseases and are valuable resources for disease diagnostic biomarkers. The most abundant proteins in plasma are high molecular weight proteins with molecular weight more than 30kDa, while the molecular weight of the most clinically significant active small proteins such as hormone, interleukin, chemokine, growth factor and interferon is below 30 kDa. If the plasma is not subjected to protein enrichment treatment, high-abundance proteins can seriously interfere with detection signals of low-molecular-weight proteins, so that low-abundance target small proteins are difficult to effectively detect, and further protein analysis with practical clinical value cannot be performed. Therefore, development of simple and efficient low molecular weight protein enrichment technology is always a core focus of plasma proteomics research. Currently, common enrichment methods of low molecular weight proteins in plasma include organic solvent precipitation, molecular cut-off ultrafiltration, solid phase extraction, sequential Precipitation Degreasing (SPD), and the like. Among them, the organic solvent precipitation method is most widely used, and acetonitrile (final concentration 60%) is generally added to plasma to precipitate high-abundance proteins, and recently, trichloroacetic acid precipitation method is also increasingly used in experimental design of low-molecular-weight proteins. The solid phase extraction method usually selects C18 or C8 as a stationary phase for enriching low molecular weight proteins and polypeptides. However, although the two methods are simple and convenient to operate, the defect of limited effect of removing the high-abundance proteins exists. The molecular cut-off ultrafiltration method separates high and low molecular weight proteins by molecular weight cut-off membrane centrifugal ultrafiltration, however, the method is easy to lose peptide fragments with molecular weight close to cut-off points, and part of lipophilic peptide fragments are easy to combine with membranes to lose. The sequential precipitation degreasing method (SPD) adopts a methyl tertiary butyl ether/methanol/water solvent system to realize sequential precipitation and degreasing, and can effectively remove high-abundance proteins, but the loss of low-molecular-weight proteins is more remarkable, and the molecular weight of most proteins in the enriched product is only 10kDa or less (10.1021/acs.jproteome.0c00232). The existing research shows that the C8 solid phase extraction fractionation method based on ammonium formate can effectively analyze low molecular weight protein (10.1016/j.mcpro.2025.101016) in cell lysate, but the specific concentration parameter of ammonium formate is not clear, and whether the method can be directly applied to enrichment of low molecular weight protein in blood plasma or not is still required to be further verified. In addition, the C18 solid phase extraction column has a larger hydrophobic surface area than the C8 solid phase extraction column, is theoretically more suitable for enriching low-abundance proteins, but has not been fully utilized in plasma low-molecular-weight protein enrichment in combination with an ammonium formate system and fractionation strategy. Therefore, it is necessary to provide a pretreatment method for plasma low molecular weight protein samples based on a C18 solid phase extraction column, which is capable of determining the concentration parameters of key reagents, and has high enrichment efficiency and low protein loss, so as to solve the defects in the prior art and improve the identification quantity and accuracy of low molecular weight proteins in plasma. Disclosure of Invention The invention aims to provide a sample pretreatment method for low-molecular-weight proteins in plasma, so as to solve the problems of low enrichment efficiency and small identification quantity of the low-molecular-weight proteins in the plasma in the prior art. In order to solve the technical problems, the invention adopts the following technical scheme: there is provided a method for sample pretreatment of low molecular weight proteins in plasma, comprising the steps of: S1, enriching low molecular weight proteins in plasma, namely preparing a C18 solid phase extraction column, washing the column with methanol, balancing the column with an ammonium formate solution, diluting the plasma with 5-20 mM ammonium formate solution, loading the diluted plasma to the C18 solid phase extraction column, washing the column with the ammonium formate solution, sequentially