Sample pretreatment method for low-molecular-weight protein in plasma

Abstract

The invention discloses a sample pretreatment method for low-molecular-weight protein in plasma, which comprises the following steps: diluting the plasma with an ammonium formate solution, loading the diluted plasma to a C18 solid-phase extraction column, sequentially carrying out gradient elution and resolution on a complex sample by using seven ACN / AmF solutions with different proportions from low to high, with the volume ratio of ACN being 15-90%, the volume ratio of AmF being 1-10%, the volume ratio of AmF being 1-10%, and the volume ratio of AmF being 1-10%. And combining subsequent reductive alkylation, enzymolysis and mass spectrometry to finally realize efficient enrichment and accurate identification of the low-molecular-weight protein in the plasma. According to the method, the experiment workload and the identification effect are balanced, the identification number and the coverage degree of the plasma low-molecular-weight protein are remarkably improved, the defects in the prior art are overcome, a more practical pretreatment means is provided for plasma proteomics research, and meanwhile due to the fact that the low-molecular-weight protein is a key biomarker resource for disease diagnosis, the application prospect is wide. The method also lays an important foundation for mining biomarkers related to diseases and assisting clinical diagnosis and medical research.

Description

Technical Field

[0001] This invention relates to the field of enrichment analysis of low molecular weight proteins in plasma, and more specifically to a sample pretreatment method for low molecular weight proteins in plasma. Background Technology

[0002] Low molecular weight proteins (LMWPs) are an important component of plasma and have been proven to be closely related to various human diseases, serving as a valuable resource for disease diagnostic biomarkers. The most abundant proteins in plasma are high molecular weight proteins with a molecular weight greater than 30 kDa, while many clinically significant active small proteins, such as hormones, interleukins, chemokines, growth factors, and interferons, have molecular weights below 30 kDa. Without plasma protein enrichment, high-abundance proteins can severely interfere with the detection signals of low-molecular-weight proteins, making it difficult to effectively detect low-abundance target small proteins and thus hindering clinically valuable protein analysis. Therefore, developing simple and efficient low-molecular-weight protein enrichment techniques remains a core focus of plasma proteomics research.

[0003] Currently, common enrichment methods for low molecular weight proteins in plasma include organic solvent precipitation, molecularly cut-off ultrafiltration, solid-phase extraction, and sequential precipitation defatting (SPD). Among these, organic solvent precipitation is the most widely used, typically involving the addition of acetonitrile (to a final concentration of 60%) to plasma to precipitate high-abundance proteins. In recent years, trichloroacetic acid precipitation has also been increasingly applied in experimental designs for low molecular weight proteins. Solid-phase extraction often uses C18 or C8 as the stationary phase for enriching low molecular weight proteins and peptides. However, while these two methods are simple to operate, they suffer from limited effectiveness in removing high-abundance proteins. Molecularly cut-off ultrafiltration separates high and low molecular weight proteins through centrifugation and ultrafiltration using a molecular weight cutoff membrane. However, this method easily loses peptides with molecular weights close to the cutoff point, and some lipophilic peptides are easily lost due to membrane binding. Sequential precipitation degreasing (SPD) uses a methyl tert-butyl ether / methanol / water solvent system to achieve sequential precipitation and degreasing. Although it can effectively remove high-abundance proteins, the loss of low molecular weight proteins is more significant. Most proteins in the enriched product have a molecular weight of only 10 kDa or less (10.1021 / acs.jproteome.0c00232).

[0004] Previous studies have shown that the C8 solid-phase extraction fractionation method based on ammonium formate can effectively analyze low molecular weight proteins in cell lysates (10.1016 / j.mcpro.2025.101016). However, this method does not specify the exact concentration parameters of ammonium formate, and its direct application to the enrichment of low molecular weight proteins in plasma requires further verification. Furthermore, while the C18 solid-phase extraction column has a larger hydrophobic surface area than the C8 column and is theoretically more suitable for enriching low-abundance proteins, it has not yet been fully utilized in the enrichment of low molecular weight proteins in plasma by combining the ammonium formate system with fractionation strategies.

[0005] Therefore, it is necessary to provide a plasma low molecular weight protein sample pretreatment method based on a C18 solid-phase extraction column, with clearly defined key reagent concentration parameters, and combining high enrichment efficiency with low protein loss, in order to overcome the shortcomings of existing technologies and improve the quantity and accuracy of low molecular weight proteins in plasma. Summary of the Invention

[0006] The purpose of this invention is to provide a sample pretreatment method for low molecular weight proteins in plasma, thereby solving the problems of low enrichment efficiency and small identification quantity of low molecular weight proteins in plasma in the prior art.

[0007] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0008] A sample pretreatment method for low molecular weight proteins in plasma is provided, comprising the following steps:

[0009] S1: Enrichment of low molecular weight proteins in plasma: A C18 solid-phase extraction column was prepared, washed with methanol, and then equilibrated with ammonium formate solution. Plasma was diluted with 5-20 mM ammonium formate solution and loaded onto the C18 solid-phase extraction column. After washing with ammonium formate solution, seven different ratios of acetonitrile / ammonium formate (ACN / AmF) solutions were used for gradient elution in order of increasing ACN volume percentage, with the ACN volume percentage ranging from 15% to 90%. The eluent was collected and dried.

[0010] S2: Enzymatic digestion of enriched proteins: The dried eluent was reconstituted with 8M urea / 100mM Tris buffer, and then subjected to TCEP reduction and IAA alkylation treatment in sequence. The urea concentration was diluted, trypsin was added for enzymatic digestion, and the reaction was terminated with formic acid to obtain peptide samples.

[0011] S3: Peptide desalting: Load the peptide sample onto a C18 desalting column, wash with 0.2% TFA / H2O, and then elute with 90% ACN / 0.2% TFA;

[0012] S4: After drying the eluent from step S3, redissolve it with 0.1% FA / H2O, and obtain mass spectrometry data by liquid chromatography-tandem mass spectrometry (LC-MS / MS). Spectronaut software is then used for library search and analysis.

[0013] Preferably, the preparation process of the C18 solid phase extraction column in step S1 includes: taking a C18 desalting column, adding a mixture of 10 mg of C18 packing and 100 μL of methanol, centrifuging at 500 rcf for 2 min to obtain a C18 solid phase extraction column with a column height of 15 mm.

[0014] Preferably, the concentration of the ammonium formate solution in step S1 is 10 mM, the pH value is 2-4, preferably pH 3.0; and the washing volume of both methanol and ammonium formate solution is 100-200 μL.

[0015] Preferably, the dilution ratio of plasma to ammonium formate solution in step S1 is 1:(8-12). Most preferably, it is 1:10.

[0016] Preferably, the different proportions of ACN / AmF solutions mentioned in step S1 are ACN / AmF solutions with a volume percentage of 15%, 30%, 35%, 37.5%, 40%, 50%, and 90% of ACN, respectively, using 10mM, pH 3.0 ammonium formate solution as the matrix, and each elution solution has a volume of 100~200μL.

[0017] Preferably, the drying conditions for the eluent in step S1 are: drying in a Speedvac at a vacuum of 0.01 mbar and a temperature of 25°C for 2 hours. The purpose is to remove acetonitrile from the eluent to avoid affecting subsequent enzymatic hydrolysis reactions.

[0018] Preferably, the pH of the 8M urea / 100mM Tris buffer in step S2 is 8.5. This is to fully reconstitute the dried protein and disrupt its higher-order structure.

[0019] Preferably, in step S2, the final concentration of TCEP is 5 mM, and the reduction reaction is carried out at room temperature for 30 min to break the disulfide bond; the final concentration of IAA is 10 mM, and the alkylation reaction is carried out in the dark for 20 min to alkylate free thiol groups.

[0020] Preferably, in step S2, the protein mixture is diluted four times with 100mM Tris buffer to reduce the urea concentration to 2M to avoid inactivation of trypsin. The mass ratio of trypsin to protein is 1:25 to 1:200, the enzymatic hydrolysis is performed overnight, and the final concentration after terminating the reaction with formic acid is 5%.

[0021] Preferably, in step S3, the centrifugation speed of the C18 desalting column is controlled at 500 rcf. After activation with 200 μL of methanol and equilibration with 200 μL of 0.2% TFA / H2O, the sample is loaded. The washing process involves washing twice with 200 μL of 0.2% TFA / H2O.

[0022] Preferably, the specific parameters for LC-MS / MS analysis in step S4 are as follows: separation is performed using an Easy nLC 1200 chromatographic system, with a ReproSil-Pur 120 C18-AQ column, mobile phase A being 0.1% FA / H2O, mobile phase B being 0.1% FA / 80% ACN, flow rate 300 nL / min, column temperature 55℃, and gradient duration 60 min; mass spectrometry data are acquired using Data-Independent Acquisition (DIA) mode, with a spray voltage of 2.1 kV, capillary temperature of 320℃, first-stage full scan resolution of 120,000, maximum injection time of 50 ms, mass scan range of 350-1500, second-stage scan resolution of 15,000, HCD collision energy of 32%, mass scan range of 200-2000, and maximum injection time of 54 ms; the Spectronaut software search parameters are: Carbamidomethyl on cysteine. The modification is fixed, the Oxidation on methionine is variable, the specific enzyme is trypsin, and the allowed number of missed cuts is 2.

[0023] Referring to Figure 1A, an ammonium formate-based C18 solid-phase extraction (AmF-C18-SPE) method was developed according to the present invention. Its operation process and working principle are as follows: First, a plasma sample is taken and passed through a C18 solid-phase extraction column. The ammonium formate system is used to enrich low molecular weight proteins. The acidic ammonium formate solution protonates the silanol groups of the C18 stationary phase, making them electrically neutral, while simultaneously giving the plasma proteins a net positive charge. Combined with the ionic strength provided by ammonium formate, electrostatic interference is shielded, allowing the proteins to be stably retained on the column through hydrophobic interactions. Subsequently, a 7... Gradient elution was performed using acetonitrile / ammonium formate (ACN / AmF) solutions with different ratios. Based on the differences in protein hydrophobicity, low concentration of acetonitrile eluted more hydrophilic proteins, medium concentration eluted more moderately hydrophobic proteins, and high concentration eluted more hydrophobic proteins, thus separating complex plasma proteins into several low molecular weight proteins and peptide components. The eluent was then collected, dried, and reconstituted. The samples were then subjected to reductive alkylation (breaking disulfide bonds and blocking free thiol groups), trypsin digestion to obtain peptides, and detected by liquid chromatography-tandem mass spectrometry (LC-MS / MS). Finally, quantification was completed using Spectronaut software for library search analysis, thereby increasing the detection quantity of low molecular weight proteins in plasma.

[0024] The core technology of this invention lies in the use of an ammonium formate-based C18 solid-phase extraction method (AmF-C18-SPE method) to enrich low molecular weight proteins in plasma. Seven ACN / AmF solutions with different ratios are selected and eluted sequentially in order of increasing ACN ratio. The eluent is then dried, and the reconstituted sample is reduced and alkylated before enzymatic hydrolysis into peptides. Finally, liquid chromatography-mass spectrometry is used for quantitative analysis, thereby achieving efficient enrichment and accurate identification of low molecular weight proteins in plasma.

[0025] One of the inventive aspects of this invention lies in the optimization of the solid-phase extraction column and the enrichment system. A C18 solid-phase extraction column, more commonly used in laboratories (with a larger hydrophobic surface area, beneficial for enriching low-abundance proteins), is selected, and an enrichment system is constructed using a 10 mM ammonium formate solution (pH 2.0–4.0, preferably pH 3.0). Acidic conditions protonate the silanol groups of the C18 stationary phase to make it electrically neutral, while simultaneously ensuring the proteins carry a net positive charge, minimizing ionic interactions between them. The ionic strength provided by ammonium formate also shields weak electrostatic interference, allowing the separation process to be based on hydrophobicity, improving experimental reproducibility, and subsequent removal via evaporation does not affect mass spectrometry analysis. Secondly, the invention also includes diluting plasma 8–12 times with ammonium formate solution, which reduces the plasma matrix effect and ensures that the proteins in the plasma are as positively charged as possible. The sample is then added to the C18 solid-phase extraction column, where protein enrichment is achieved through hydrophobic interactions between the protein and the stationary phase.

[0026] The key inventive point of this invention lies in the optimization of the elution gradient. Firstly, this invention establishes a stepwise elution using 7 gradients of acetonitrile / ammonium formate (ACN / AmF) solutions. Reducing the number of fractions (e.g., 4) or reversing the gradient order would decrease the number of proteins identified, while increasing the number of fractions (e.g., 8 or more) would significantly increase the workload, making it counterproductive. Research has found that the 7 gradients of acetonitrile / ammonium formate (ACN / AmF) solutions can elute proteins sequentially according to their hydrophobicity. Specifically, low concentrations (below 30%) of acetonitrile elute strongly hydrophilic proteins, medium concentrations (30%-40%) elute the most abundant moderately hydrophobic proteins, and high concentrations (above 40%) elute strongly hydrophobic proteins. This approach breaks down complex samples into simpler components, reducing the complexity of subsequent enzymatic peptide digestion and improving the accuracy of quantifying low-abundance peptides.

[0027] The present invention then compared the grading effects of grading scheme one (15% ACN / AmF solution, 30% ACN / AmF solution, 35% ACN / AmF solution, 37.5% ACN / AmF solution, 40% ACN / AmF solution, 50% ACN / AmF solution, 90% ACN / AmF solution) and grading scheme two (15% ACN / AmF solution, 20% ACN / AmF solution, 25% ACN / AmF solution, 30% ACN / AmF solution, 35% ACN / AmF solution, 40% ACN / AmF solution, 75% ACN / AmF solution).

[0028] The SDS-PAGE analysis results of this invention show that the seven fractionation significantly reduces the complexity of plasma proteins, and low molecular weight proteins appear clearly on the gel image, indicating that this method can effectively enrich low molecular weight proteins. Each eluted fraction in fractionation scheme one exhibits a relatively high number of proteins (e.g., ...). Figure 1 As shown in Figure B), the protein bands eluted by the 20% ACN / AmF solution and 25% ACN / AmF solution in fractionation scheme two were significantly fewer than those of the other components (e.g., ...). Figure 1 As shown in C), this leads to low separation efficiency and poor total number of identifications.

[0029] Therefore, the present invention ultimately determined that the first elution scheme (15% ACN / AmF solution, 30% ACN / AmF solution, 35% ACN / AmF solution, 37.5% ACN / AmF solution, 40% ACN / AmF solution, 50% ACN / AmF solution, 90% ACN / AmF solution) was the most preferred elution scheme.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] The fractionation using 7 gradient acetonitrile / ammonium formate eluents significantly reduced the complexity of plasma proteins, and low molecular weight proteins were clearly presented in the SDS-PAGE gel images. Compared with sequential precipitation defatting (SPD), acetonitrile precipitation (ACN), and traditional solid phase extraction (SPE), the method of this invention (AmF-C18-SPE) can enrich more low molecular weight proteins.

[0032] The method of this invention identifies approximately 2,000 proteins, far exceeding the number identified by untreated plasma samples and other enrichment methods. Although proteins with fewer than 100 amino acids account for only 6%, their absolute number is significantly higher than that of other methods. Furthermore, the identified proteins essentially cover the low molecular weight proteins enriched by other methods, effectively improving the depth of plasma low molecular weight protein identification, which is of great significance for the discovery of biomarkers for disease diagnosis.

[0033] In summary, this invention addresses the shortcomings of existing methods for enriching low molecular weight proteins (LMW) in plasma, such as significant interference from high-abundance proteins, substantial loss of LMW proteins, and limited identification quantity. It develops a C18 solid-phase extraction method based on ammonium formate. This method achieves controllable enrichment through hydrophobic interactions by using the ammonium formate system, reduces matrix effects by diluting plasma, and elutes complex samples using seven gradient acetonitrile / ammonium formate solutions. Combined with subsequent reductive alkylation, enzymatic digestion, and mass spectrometry analysis, it efficiently enriches and improves the detection and identification of LMW proteins in plasma. This method balances experimental workload with identification efficiency, significantly increasing the identification quantity (approximately 2000) and coverage of LMW proteins in plasma. It also overcomes the shortcomings of existing technologies, providing a more practical pretreatment method for plasma proteomics research. Furthermore, since LMW proteins are key biomarker resources for disease diagnosis, this method also lays an important foundation for the discovery of disease-related biomarkers and for assisting clinical diagnosis and medical research. Attached Figure Description

[0034] Figure 1 This invention provides a preliminary detection correlation spectrum for enriching low molecular weight proteins in plasma using a C18 solid-phase extraction method based on ammonium formate; wherein,

[0035] A demonstrates the operational procedure of the C18 solid-phase extraction method based on ammonium formate;

[0036] Image B is an SDS-PAGE electrophoresis analysis of the fractions eluted by the first gradient scheme, showing plasma proteins and low molecular weight proteins in the fractions eluted by different gradients (15%, 30%, 35%, 37.5%, 40%, 50%, 90% acetonitrile / ammonium formate solution);

[0037] C is an SDS-PAGE electrophoresis analysis of the fractions eluted by the two gradient scheme, showing plasma proteins and low molecular weight proteins in the fractions eluted by different gradients (15%, 20%, 25%, 30%, 35%, 40%, 75% acetonitrile / ammonium formate solution);

[0038] D is an SDS-PAGE electrophoresis analysis of low molecular weight proteins obtained by classical enrichment methods (sequential precipitation defatting, acetonitrile precipitation, and traditional solid-phase extraction). Each method was performed in triplicate.

[0039] E shows the protein distribution identified by untreated plasma, sequential precipitation defatting, acetonitrile precipitation, conventional solid-phase extraction, and AmF-C18-SPE method. The percentages in the figure represent the proportion of proteins with fewer than 100 amino acids in the total number of proteins identified by each method.

[0040] F is an analysis diagram of protein overlap, showing the overlap between proteins identified by untreated plasma, sequential precipitation defatting, acetonitrile precipitation, conventional solid-phase extraction, and the AmF-C18-SPE method. Detailed Implementation

[0041] The present invention will be further described below with reference to specific embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, the techniques used in the embodiments are conventional practices in the art, or experimental methods recommended by the reagent kit and instrument manufacturers. Unless otherwise specified, the reagents and materials used in the embodiments are commercially available.

[0042] Example 1: A method for enriching low molecular weight proteins in plasma using ammonium formate-based C18 solid-phase extraction (AmF-C18-SPE).

[0043] I. Experimental Materials and Reagents

[0044] Solid phase extraction column related materials: C18 desalting column (supplier: Shanghai Omicsolution Co., Ltd., catalog number: OSFP0010); C18 packing material (Waters, 80μm); laboratory-made C18 analytical column (particle size 1.9μm, 75μm×25cm, brand: ReproSil-Pur 120 C18-AQ).

[0045] Reagents: Methanol (MeOH), acetonitrile (ACN), formic acid (FA), trifluoroacetic acid (TFA), ammonium formate (AmF), urea, tris(2-carboxyethyl)phosphine (TCEP), iodoacetamide (IAA), trypsin, and Tris were all of chromatographic or mass spectrometry grade; the water used in the experiment was ultrapure water.

[0046] Solution preparation:

[0047] 10 mM ammonium formate solution (AmF, pH 3.0);

[0048] Fractionation scheme 1: 15% ACN / AmF solution, 30% ACN / AmF solution, 35% ACN / AmF solution, 37.5% ACN / AmF solution, 40% ACN / AmF solution, 50% ACN / AmF solution, and 90% ACN / AmF solution (all using 10mM, pH 3.0 ammonium formate solution as the matrix, with acetonitrile volume percentages ranging from 15% to 90%).

[0049] Classification scheme 2: 15% ACN / AmF solution, 20% ACN / AmF solution, 25% ACN / AmF solution, 30% ACN / AmF solution, 35% ACN / AmF solution, 40% ACN / AmF solution, 75% ACN / AmF solution;

[0050] 8M urea / 100mM Tris buffer (pH 8.5);

[0051] 0.2% TFA / H2O solution, 90% ACN / 0.2% TFA solution;

[0052] 0.1% FA / H2O solution, 0.1% FA / 80% ACN solution (LC-MS / MS mobile phase).

[0053] II. Experimental Methods

[0054] (I) Enrichment and processing of low molecular weight proteins in plasma using the AmF-C18-SPE method

[0055] 1. Laboratory-made C18 solid-phase extraction column: Take a C18 desalting column, weigh 10 mg of C18 packing material, add 100 μL of methanol and mix well. Then add the mixture to the desalting column using a pipette. Centrifuge at 500 rcf for 2 min to obtain a C18 solid-phase extraction column with a column height of 15 mm.

[0056] 2. Plasma sample enrichment:

[0057] Solid-phase extraction column pretreatment: first wash and activate with 200 μL methanol, then equilibrate twice with 200 μL 10 mM ammonium formate solution (pH 3.0);

[0058] Sample loading: Take 20 μL of plasma sample, add 200 μL of 10 mM ammonium formate solution to dilute, mix thoroughly and load the sample into the pretreated extraction column;

[0059] Washing and elution: The extraction column was washed twice with 200 μL of 10 mM ammonium formate solution, and then eluted stepwise with 7 gradient elution solutions (100 μL each) of fractionation scheme 1 and fractionation scheme 2 respectively, and 7 eluents were collected for each scheme.

[0060] Drying: Seven portions of each of the two fractionation schemes were placed in Speedvac and dried for 2 hours under a vacuum of 0.01 mbar and a temperature of 25°C to remove the solvent.

[0061] 3. Enzymatic digestion of enriched proteins:

[0062] Reconstitution: The dried eluent was fully reconstituted with 50 μL of 8M urea / 100mM Tris buffer (pH 8.5); half of the reconstituted sample was used for SDS-PAGE analysis to verify the enrichment effect of low molecular weight proteins.

[0063] Reduction and alkylation: TCEP was added to the remaining complex solution to a final concentration of 5 mM, and the reaction was carried out at room temperature for 30 min to reduce disulfide bonds; then IAA was added to a final concentration of 10 mM, and the reaction was carried out in the dark for 20 min to alkylate free thiol groups;

[0064] Enzymatic hydrolysis: Dilute the protein mixture 4 times with 100mM Tris buffer (to reduce the urea concentration to 2M), add trypsin at a trypsin to protein ratio of 1:50 (w / w), and hydrolyze overnight; the next day, add formic acid to a final concentration of 5% to terminate the enzymatic hydrolysis reaction and obtain peptide samples.

[0065] 4. Peptide desalting:

[0066] Desalting column pretreatment: Take a C18 desalting column, centrifuge at 500 rcf, activate it with 200 μL of methanol, and then equilibrate it with 200 μL of 0.2% TFA / H2O solution;

[0067] Sample loading and washing: Add the enzymatically digested peptide sample to the desalting column and wash twice with 200 μL of 0.2% TFA / H2O solution;

[0068] Elution and drying: Elute the peptides with 90% ACN / 0.2% TFA solution, collect the eluent, and dry it in Speedvac for later use.

[0069] 5. SDS-PAGE analysis:

[0070] For the proteins enriched by the two grading methods, 50 μL of 8M urea / 100mM Tris buffer (pH 8.5) was added to reconstitute the proteins. 15 μL of each elution buffer was taken from each elution step, mixed with the loading buffer, and then analyzed by SDS-PAGE to observe the enrichment of low molecular weight proteins in plasma.

[0071] The results are shown in Figures B and C in Figure 1:

[0072] Figure 1B shows the protein distribution under fractionation scheme one (elution gradient of 15%, 30%, 35%, 37.5%, 40%, 50%, 90% ACN / AmF solution). Lane 1 is the unfractionated plasma sample, and lanes 2-8 correspond to the seven elution fractions of fractionation scheme one. It can be seen that each elution fraction shows abundant protein bands. The protein signals in the low molecular weight region (10-35kDa) are clear and evenly distributed, indicating that this gradient can fully separate low molecular weight proteins with different hydrophobicities, and each fraction is enriched with a large amount of target protein.

[0073] In Figure 1, corresponding to fractionation scheme 2 (elution gradient of 15%, 20%, 25%, 30%, 35%, 40%, 75% ACN / AmF solution), the protein bands in lane 2 (elution with 20% ACN / AmF) and lane 3 (elution with 25% ACN / AmF) are significantly sparse, with a large difference in band abundance compared to other components. This indicates that the protein enrichment of some components under this gradient is insufficient and the separation efficiency is low.

[0074] In summary, the gradient design of fractionation scheme one is more reasonable and can ensure the protein abundance of each eluted component, laying the foundation for increasing the total number of low molecular weight proteins identified subsequently. Therefore, fractionation scheme one (15% ACN / AmF solution, 30% ACN / AmF solution, 35% ACN / AmF solution, 37.5% ACN / AmF solution, 40% ACN / AmF solution, 50% ACN / AmF solution, 90% ACN / AmF solution) is determined to be the optimal elution scheme of this invention.

[0075] 6. LC-MS / MS Analysis:

[0076] Sample reconstitution: The dried peptide fragments were reconstituted with 10 μL of 0.1% FA / H2O solution, centrifuged at 20000 rcf for 15 min, and 7 μL of the supernatant was transferred into a chromatographic sample vial;

[0077] Chromatographic separation: 2 μL of sample was separated using an Easy nLC 1200 chromatographic system. The column was a self-made laboratory product with a particle size of 1.9 μm and dimensions of 75 μm × 25 cm. Brand: ReproSil-Pur 120 C18-AQ. Mobile phase A was 0.1% FA / H2O, and mobile phase B was 0.1% FA / 80% ACN. Flow rate: 300 nL / min, column temperature: 55℃, gradient duration: 60 min, 0 min: 2%, 1 min: 12%, 51 min: 45%, 54 min: 90%, 56 min: 100%, 60 min: 100%.

[0078] Mass spectrometry detection: Mass spectrometry data were acquired using the DIA mode of a Thermo Scientific Orbitrap Eclipse mass spectrometer with the following parameters: spray voltage 2.1 kV, capillary temperature 320 °C, first-stage full scan resolution 120,000, maximum injection time 50 ms, mass scan range 350-1500, second-stage scan resolution 15,000, HCD collision energy 32%, mass scan range 200-2000, and maximum injection time 54 ms.

[0079] 7. Data Analysis:

[0080] The collected DIA data were analyzed using Spectronaut software. The Homo sapiens FASTA file was downloaded from the Uniprot database (reviewed). The main search parameters were as follows: Carbamidomethyl modification on cysteine ​​was set to fixed, and Oxidation on methionine was set to variable. Trypsin was selected for specific enzyme digestion, with an allowable number of missed digestions of 2.

[0081] The number of proteins identified in each gradient of the grading scheme is shown in Table 1 below.

[0082] Table 1. Number of proteins identified by the seven gradient elutions in gradation scheme one.

[0083] solutions used for fractionation Number of peptides identified Number of protein identifications Elution 1 10809 1355 Elution 2 12514 1817 Elution 3 12049 1756 Elution 4 11138 1638 Elution 5 10263 1506 Elution 6 11105 1526 Elution 7 7445 1076 total 15828 2085

[0084] Example 2: Comparison of the AmF-C18-SPE method with existing technologies (sequential precipitation defatting, acetonitrile precipitation, and traditional solid-phase extraction).

[0085] To demonstrate the advantages of this invention in identifying low molecular weight proteins, this embodiment employs commonly used low molecular weight protein enrichment methods to analyze low molecular weight proteins in plasma, and also analyzes proteins in untreated plasma samples.

[0086] Sequential precipitation defatting (SPD): 20 μL of plasma was mixed with 20 μL of H₂O and 100 μL of methyl tert-butyl ether by vortexing. Then, 60 μL of methanol was added and vortexed again. The mixture was incubated at 4°C for 30 min to precipitate. After centrifugation at 20,000 rcf at 4°C for 20 min, the supernatant was collected and mixed with 200 μL of methyl tert-butyl ether and 40 μL of H₂O by vortexing. The mixture separated into two phases. After centrifugation at 1,000 rcf at 4°C for 10 min, the lower layer was collected and dried on a Speedvac.

[0087] Acetonitrile precipitation method (ACN): 20 μL of plasma was diluted with 60 μL of H2O, then 120 μL of ACN was added and vortexed. The mixture was then placed in a 4°C refrigerator for 1 h, followed by centrifugation at 20000 rcf at 4°C for 20 min. The supernatant was collected and finally dried on a Speedvac.

[0088] Traditional solid-phase extraction (SPE): 20 μL of plasma was diluted with 200 μL of 0.2% TFA / H2O. The C18 desalting column was centrifuged at 500 rcf. The column was activated with 200 μL of methanol and equilibrated with 200 μL of 0.2% TFA / H2O. The diluted plasma sample was then added to the desalting column. The sample was washed with 200 μL of 0.2% TFA / H2O, repeated once, eluted with 90% ACN / 0.2% TFA, and finally dried on a Speedvac.

[0089] For the proteins enriched by these three methods, 50 μL of 8M urea / 100mM Tris buffer (pH 8.5) was added for reconstitution. 5 μL of the SPD method, 2 μL of the ACN method, and 2 μL of the SPE method were taken and analyzed by SDS-PAGE to observe the enrichment of low molecular weight proteins in plasma.

[0090] For the SPD method, take 35 μL; for the ACN precipitation method, take 20 μL; and for the SPE method, take 20 μL. Take 0.1 μL of plasma and dilute it with 20 μL of 8M urea / 100mM Tris buffer (pH 8.5) before enzymatic digestion. The procedures for TCEP reduction, IAA alkylation, overnight digestion, and peptide desalting are the same as described above. The dried peptides are reconstituted with 10 μL of 0.1% FA / H2O, centrifuged at 20000 rcf for 15 min, and 7 μL of the supernatant is added to a chromatographic vial for later use. The LC-MS / MS analysis conditions and Spectronaut software library search parameters are the same as described above. Data on the enrichment methods and the number of plasma samples identified are shown in Table 2 below.

[0091] Table 2. Number of proteins identified by plasma, SPD method, ACN precipitation method, SPE method, and AmF-C18-SPE method.

[0092] Number of peptides identified Number of protein identifications Untreated plasma 5302 428 SPD method enrichment 1438 313 ACN precipitation enrichment 4719 646 SPE enrichment method 7329 993 AmF-C18-SPE method 15828 2085

[0093] SDS-PAGE verification results are as follows Figure 1 As shown in Figure D: All seven eluted fractions obtained using the AmF-C18-SPE method exhibited abundant protein bands in the gel image, with clear signals for low molecular weight proteins; the results of protein identification using several enrichment methods are as follows... Figure 1 As shown in Figures E and F, compared with the SPD, ACN, and SPE methods, the method of this invention enriches more types of low molecular weight proteins and effectively reduces sample complexity, indicating that the method of this invention has great application prospects in clinical diagnosis.

[0094] The above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of the invention. Various variations can be made to the above embodiments of the present invention. All simple and equivalent changes and modifications made in accordance with the claims and description of this application fall within the protection scope of the claims of this patent. All aspects not described in detail in this invention are conventional technical content.

Claims

1. A sample pretreatment method for low molecular weight proteins in plasma, characterized in that, Includes the following steps: S1: Enrichment of low molecular weight proteins in plasma: A C18 solid-phase extraction column was prepared, washed with methanol, and then equilibrated with ammonium formate solution. Plasma was diluted with 5-20 mM ammonium formate solution and loaded onto the C18 solid-phase extraction column. After washing with ammonium formate solution, seven different ratios of ACN / AmF solution were used for gradient elution in order of ACN volume percentage from low to high, with the ACN volume percentage between 15% and 90%. The eluent was collected and dried. S2: Enzymatic digestion of enriched proteins: The dried eluent was reconstituted with 8M urea / 100mM Tris buffer, and then subjected to TCEP reduction and IAA alkylation treatment in sequence. The urea concentration was diluted, trypsin was added for enzymatic digestion, and the reaction was terminated with formic acid to obtain peptide samples. S3: Peptide desalting: Load the peptide sample onto a C18 desalting column, wash with 0.2% TFA / H2O, and then elute with 90% ACN / 0.2% TFA; S4: After drying the eluent from step S3, redissolve it with 0.1% FA / H2O, obtain mass spectrometry data by LC-MS / MS analysis, and use Spectronaut software for library search analysis.

2. The sample pretreatment method according to claim 1, characterized in that, The C18 solid-phase extraction column mentioned in step S1 is prepared in the laboratory. The preparation process includes: taking a C18 desalting column, adding a mixture of 10 mg of C18 packing and 100 μL of methanol, centrifuging at 500 rcf for 2 min to obtain a C18 solid-phase extraction column with a column height of 15 mm.

3. The sample pretreatment method according to claim 1, characterized in that, The concentration of the ammonium formate solution in step S1 is 10 mM, and the pH value is 2-4; the washing volume of both methanol and ammonium formate solution is 100-200 μL.

4. The sample pretreatment method according to claim 1, characterized in that, In step S1, the dilution ratio of plasma to ammonium formate solution is 1:(8-12).

5. The sample pretreatment method according to claim 1, characterized in that, The different proportions of ACN / AmF solutions mentioned in step S1 are ACN / AmF solutions with a volume percentage of 15%, 30%, 35%, 37.5%, 40%, 50%, and 90% respectively, using 10mM, pH 3.0 ammonium formate solution as the matrix. The volume of each elution solution is 100~200μL.

6. The sample pretreatment method according to claim 1, characterized in that, The drying conditions for the eluent in step S1 are as follows: drying in Speedvac at a vacuum of 0.01 mbar and a temperature of 25°C for 2 hours.

7. The sample pretreatment method according to claim 1, characterized in that, In step S2, the pH of the 8M urea / 100mM Tris buffer is 8.5; the final concentration of TCEP is 5mM, and the reduction reaction is carried out at room temperature for 30 min; the final concentration of IAA is 10mM, and the alkylation reaction is carried out in the dark for 20 min.

8. The sample pretreatment method according to claim 1, characterized in that, In step S2, the protein mixture was diluted four times with 100mM Tris buffer. The mass ratio of trypsin to protein was 1:25 to 1:

200. The enzymatic digestion was performed overnight, and the final concentration after terminating the reaction with formic acid was 5%.

9. The sample pretreatment method according to claim 1, characterized in that, In step S3, the centrifugation speed of the C18 desalting column is controlled at 500 rcf. After activation with 200 μL of methanol and equilibration with 200 μL of 0.2% TFA / H2O, the sample is loaded. The washing process is two washes with 200 μL of 0.2% TFA / H2O.

10. The sample pretreatment method according to claim 1, characterized in that, The specific parameters for LC-MS / MS analysis in step S4 are as follows: Separation was performed using an Easy nLC 1200 chromatographic system with a ReproSil-Pur 120 C18-AQ column. Mobile phase A was 0.1% FA / H2O, mobile phase B was 0.1% FA / 80% ACN, flow rate was 300 nL / min, column temperature was 55℃, and gradient duration was 60 min. Mass spectrometry data were acquired using DIA mode with a spray voltage of 2.1 kV, capillary temperature of 320℃, first-stage full scan resolution of 120,000, maximum injection time of 50 ms, mass scan range of 350-1500, second-stage scan resolution of 15,000, HCD collision energy of 32%, mass scan range of 200-2000, and maximum injection time of 54 ms. The Spectronaut software search parameters were: Carbamidomethyl modification on cysteine ​​was fixed, and Oxidation on methionine was... It is a variable modification, the specific enzyme is trypsin, and the allowed number of missed cuts is 2.

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