Integral centrifugal column for enriching S-nitrosopeptide as well as preparation method and application of integral centrifugal column

Abstract

The invention discloses an integral centrifugal column for enriching S-nitrosopeptide as well as a preparation method and application of the integral centrifugal column, and belongs to the technical field of SNO proteomics analysis. The method disclosed by the invention comprises the following steps: adding an integral material pre-polymerization solution into a closed blank centrifugal column, and carrying out in-situ thermal initiation polymerization reaction to obtain a substrate integral material centrifugal column; and swelling the substrate integral material centrifugal column, adding a phosphate buffer solution of pyridine dithio ethylamine hydrochloride, standing and cleaning to obtain the integral material centrifugal column for enriching the SNO peptide. The centrifugal column can realize integrated reduction, marking and enrichment of SNO peptides, and is applied to SNO proteomics analysis in complex biological samples. The method solves the technical problem that the existing method is difficult to simultaneously reduce / mark / enrich SNO protein or peptide in a complex sample, effectively reduces the loss and degradation of the SNO peptide in the sample treatment process through the integrated sample treatment process, and obviously improves the identification scale of the SNO protein and the modification site.

Description

Technical Field

[0001] This invention belongs to the field of SNO proteomics analysis technology, specifically relating to a monolithic centrifuge column for enriching S-nitrosyl peptides, its preparation method, and its application. Background Technology

[0002] S-nitrosylation (SNO) is an important and reversible post-translational modification of proteins, in which nitric oxide (NO) or related nitrosylating agents selectively and covalently attach a nitrosyl group (-NO) to the thiol group of a protein's cysteine ​​residue, ultimately forming an S-nitrosothiol. This modification can significantly affect protein structure, activity, stability, and intracellular localization, thus playing a crucial role in various physiological and pathological processes, including cell signal transduction, immune regulation, and oxidative stress responses. Abnormal SNO expression is closely related to the pathogenesis of various diseases, such as tumors, neurodegenerative diseases, cardiovascular diseases, and inflammatory responses. Therefore, in-depth research on SNO-modified proteins and their related pathways is of great significance for understanding signal transduction mechanisms under physiological and pathological conditions and for developing potential drugs.

[0003] Proteomics strategies based on liquid chromatography-tandem mass spectrometry (LC-MS / MS) enable high-throughput identification and quantitative analysis of SNO proteins and their modification sites. However, large-scale SNO proteomics analysis via direct injection faces significant challenges due to the extremely low abundance of endogenous SNO modifications and the instability of the SN bonds in SNO modifications.

[0004] To address these limitations in SNO proteomics analysis, researchers have developed various techniques for the specific labeling and enrichment of SNO proteins or peptides from complex biological samples prior to mass spectrometry analysis. The most widely used technique is the biotin-switching (BST) technique proposed by Jaffrey et al. in 2001. This strategy achieves the detection and analysis of SNO proteins or peptides by sequentially blocking free thiol groups, selectively reducing SNO groups, labeling newly generated thiol sites, and finally performing affinity capture. In recent years, researchers have also developed various other chemical probes and corresponding solid-phase enrichment materials for labeling SNO-reduced thiol groups, such as His-tag with nickel columns, ICAT with avidin gels, cysTMT or iodoTMT with anti-TMT resins, CysPAT with TiO2 nanoparticles, FIAM with fluorinated graphene, and IAA-alkyne with cys-BOOST. Furthermore, chemical probes SNOTRAP and biotin-SO2H have been developed for the direct labeling of SNO groups, followed by capture of SNO proteins or peptides using streptavidin magnetic beads.

[0005] However, the aforementioned techniques all rely on stepwise probe labeling and affinity enrichment processes, which are time-consuming and labor-intensive. The complex sample processing steps also increase the risk of SNO protein loss. To reduce the impact of these steps, some studies have immobilized reactive groups on solid-phase supports, thereby achieving simultaneous labeling and capture of target SNO proteins. However, existing solid-phase methods still have significant limitations. For example, while gold nanoparticles exhibit good reactivity, they are prone to aggregation during enrichment and lack systematic validation in complex biological matrices; organomercury resins, although effective at capturing thiol groups, are limited by biosafety concerns due to mercury toxicity; SNO protein resin-assisted capture technology (SNO-RAC), while possessing high specificity and efficiency, suffers from non-specific adsorption due to the high hydrophilicity of traditional resins, affecting the binding efficiency and identification coverage of SNO peptides. Furthermore, most current SNO enrichment strategies are conducted in SNO systems induced by exogenous NO donors, and research truly targeting endogenous SNO proteins and modification sites remains very limited. Given the above limitations, developing new solid-phase carriers that can be used to simultaneously label and enrich SNO proteins or peptides in complex biological samples is of great significance for exploring the biological functions and pathological effects of SNO. Summary of the Invention

[0006] The purpose of this invention is to provide a monolithic centrifuge column for enriching S-nitrosyl peptides, its preparation method, and its application. This invention solves the technical problem that existing methods are difficult to simultaneously reduce / label / enrich SNO proteins or peptides in complex samples. Through an integrated sample processing process, the loss and degradation of SNO peptides during sample processing are effectively reduced, and the identification scale of SNO proteins and modification sites is significantly improved.

[0007] To achieve the above objectives, the present invention employs the following technical solution: This invention discloses a method for preparing a monolithic centrifuge column for enriching S-nitrosyl peptides, comprising the following steps: The monolithic material prepolymer solution was added to a pre-sealed blank polypropylene centrifuge column for thermally initiated in-situ polymerization. After the reaction was completed, the monolithic substrate centrifuge column was obtained. After swelling the monolithic substrate centrifuge column, phosphate buffer of pyridine dithioethylamine hydrochloride was added, followed by standing and washing to obtain a monolithic centrifuge column enriched with S-nitrosotriates.

[0008] Further, by mass percentage, the prepolymer solution of the monolithic material comprises 10%~15% N-acryloyloxysuccinimide, 20%~30% ethylene glycol dimethacrylate, 1%~3% octavinyl polyhedral oligomeric silsesquioxane, 55%~65% toluene and azobisisobutyronitrile; The content of the azobisisobutyronitrile is 1% to 2% of the total mass of N-acryloyloxysuccinimide and octavinyl polyhedral oligomeric silsesquioxane.

[0009] Furthermore, the method for preparing the monolithic material prepolymer solution is as follows: N-acryloyloxysuccinimide, ethylene glycol dimethacrylate, octavinyl polyhedral oligomeric silsesquioxane and toluene were mixed, degassed by ultrasound, and fully dissolved at 65-75 °C. Then, azobisisobutyronitrile was added and mixed to obtain a prepolymer solution of the whole material.

[0010] Further, 150-300 μL of prepolymer solution of the whole material was added to an 800 μL polypropylene centrifuge column.

[0011] Furthermore, the temperature of the thermally initiated in-situ polymerization reaction is 65~75 °C, and the reaction time is 12~24 h.

[0012] Furthermore, after the substrate monolithic material centrifuge column is swollen with acetonitrile, it is equilibrated with phosphate buffer, and then phosphate buffer of pyridine dithioethylamine hydrochloride is added to the substrate monolithic material; the concentration of the phosphate buffer is 50~100 mM and the pH value is 7.4~8.2.

[0013] Further, in the phosphate buffer solution of pyridine dithioethylamine hydrochloride, the concentration of pyridine dithioethylamine hydrochloride is 0.5~2 M; and the volume of the phosphate buffer solution of pyridine dithioethylamine hydrochloride is 200~800 μL. The placement temperature is 2~6 ℃, and the time is 2~4 h.

[0014] The present invention also discloses a monolithic centrifuge column for enriching S-nitrosyl peptides prepared by the above preparation method.

[0015] This invention also discloses the above-mentioned monolithic centrifugation column for enriching S-nitrosyl peptides in the integrated reduction, labeling, enrichment of S-nitrosyl peptides and its application in S-nitrosyl proteomics analysis of complex biological samples (such as cells, animal plasma / serum / tissue, clinically collected samples, etc.). The specific steps for S-nitroso proteomics analysis are as follows: After alkylating the protein sample dilution solution, pre-cooled acetone was added to precipitate the protein. The protein precipitate was then collected by centrifugation to obtain the alkylated protein precipitate. The alkylated protein precipitate was resuspended in HEPES buffer, and then incubated with trypsin to obtain the enzymatically digested peptide solution. Sodium ascorbate was added to the peptide solution, and then added to a swollen and equilibrated monolithic centrifuge column for enriching S-nitrosyl peptides. The column was then placed at room temperature to perform the integrated reduction, labeling, and enrichment of S-nitrosyl peptides. Elution solution was added to the whole centrifuge column containing the S-nitrosyl peptide sample, and the column was placed at room temperature to elute the S-nitrosyl peptide. After elution, the peptide samples were desalted and analyzed by liquid chromatography-mass spectrometry to identify S-nitroso proteins and modification sites.

[0016] The protein sample dilution buffer is prepared by diluting the protein sample to 0.5-2 mg / mL with HEPES buffer containing 10-40 mM iodoacetamide; the concentration of the HEPES buffer is 50-100 mM and the pH is 7.2-7.8; the alkylation reaction is carried out at room temperature and in the dark for 30-60 min. The temperature of the pre-cooled acetone is -15 to -20°C; the volume ratio of acetone to protein sample dilution is (3 to 5): 1. The mass ratio of trypsin to protein precipitate is 1:20 to 1:50; The final concentration of sodium ascorbate is 10-20 mM; the integrated reduction, labeling, and enrichment of S-nitrosotropic peptides are carried out by placing the product at room temperature for 1-2 h. The elution solution is a TCEP or DTT solution with a concentration of 5-20 mM, and the elution time is 0.5-2 h.

[0017] Furthermore, after the integrated reduction, labeling, and enrichment of S-nitrosolated peptides, the monolithic centrifugal column for enriching S-nitrosolated peptides is regenerated by adding 200-800 μL of methanol solution containing 0.5-2 M diphenyl disulfide and placing it at room temperature for 1-5 h to restore it to its initial state for reuse. The number of times the device is reused is no less than 6.

[0018] Compared with the prior art, the present invention has the following beneficial effects: This invention discloses a method for preparing a monolithic centrifuge column for enriching S-nitrosolated peptides (SNO peptides). During the preparation process, the active reactive group pyridine disulfide is fixed onto a solid monolithic material. This monolithic material is porous, has a large specific surface area, and is hydrophobic. Therefore, after ascorbic acid selectively reduces SNO to generate thiol groups, these groups can immediately react with the pyridine disulfide groups to achieve site labeling. Because the pyridine disulfide groups are bound to the solid material, solid-phase enrichment is simultaneously achieved. N-acryloyloxysuccinimide (NAS) is used as the functional monomer, octavinyl polyhedral oligomeric silsesquioxane (OVPOS) and ethylene glycol dimethacrylate (EDMA) are used as hybrid crosslinking agents, toluene is used as the porogen, and azobisisobutyronitrile (AIBN) is used as the initiator. A three-dimensional porous monolithic material is directly constructed within the centrifuge column using in-situ thermally initiated polymerization. The active NHS ester groups provided by NAS can specifically react with primary amines under mild conditions, achieving efficient covalent modification of the pyridine disulfide groups. Meanwhile, the multi-functional pyridine disulfide groups of OVPOS... The branched structure significantly increases the exposure of reaction sites and optimizes pore structure and permeability while ensuring the mechanical strength of the material. This allows the ascorbic acid-mediated SNO reduction process and the thiol-disulfide bond exchange reaction to occur simultaneously within the same column. As a result, the monolithic centrifuge column for enriching S-nitrosyl peptides prepared by this invention can achieve integrated reduction / labeling / enrichment of SNO peptides within the column. The SNO-MSC preparation process is simple, reproducible, and regenerable, which is helpful for high-throughput analysis in practical applications. At the same time, it effectively reduces costs and improves resource utilization.

[0019] This invention also discloses the application of the aforementioned monolithic centrifuge column for enriching S-nitrosylated peptides. This column enables integrated reduction / labeling / enrichment of SNO peptides within the column, facilitating a simplified SNO proteomics analysis workflow. This method integrates multiple steps into a single operation, reducing transfer steps and shortening processing time, significantly optimizing the sample processing flow. This workflow effectively reduces sample loss and degradation during pretreatment, thereby improving the identification efficiency and coverage of SNO peptides and reducing false positives.

[0020] Furthermore, based on relevant experimental results, the SNO proteomics analysis workflow developed in this invention was applied to the identification of endogenous SNO proteins and modification sites in five organ tissues of mice, and the identification results were significantly better than those of previously developed methods. This result fully demonstrates that the newly developed workflow of this invention has extremely high enrichment efficiency and broad detection coverage, providing a reliable and efficient tool for large-scale SNO proteomics analysis. Attached Figure Description

[0021] Figure 1 This is a schematic diagram of the preparation process of the monolithic centrifugal column for enriching S-nitrosyl peptides according to the present invention; Figure 2To ensure the batch-to-batch and batch-to-batch reproducibility of the monolithic centrifuge column for enriching S-nitrosyl peptides according to the present invention; Where: A, n = 4 pillars; B, n = 3 batches; Figure 3 To ensure the reusability and storage stability of the monolithic centrifugal column for enriching S-nitrosyl peptides in this invention after regeneration. Among them: A. repeatability; B. stability; Figure 4 A flowchart for SNO proteomics analysis in complex biological samples using the monolithic centrifuge column enriched with S-nitrosyl peptides of the present invention. Figure 5 The results of the enrichment efficiency and selectivity evaluation of SNO protein / peptide in mouse liver samples; Among them: A. Representative total ion chromatography; B. Total peptide / protein count and SNO peptide / protein count; Figure 6 This refers to the number of SNO proteins and SNO modification sites identified in wild-type mouse tissues (including heart, liver, lung, kidney, and brain) after integrated intracolumn labeling and enrichment by SNO-MSC. Detailed Implementation

[0022] To enable those skilled in the art to understand the features and effects of the present invention, the terms and expressions used in the specification and claims are explained and defined in general below. Unless otherwise specified, all technical and scientific terms used herein have the ordinary meaning understood by those skilled in the art regarding the present invention, and in case of conflict, the definitions in this specification shall prevail.

[0023] The theories or mechanisms described and disclosed herein, whether right or wrong, should not in any way limit the scope of the invention, that is, the contents of the invention can be implemented without being limited by any particular theory or mechanism.

[0024] In this document, all features defined by numerical ranges or percentage ranges, such as numerical values, quantities, contents, and concentrations, are for the sake of brevity and convenience only. Accordingly, descriptions of numerical ranges or percentage ranges should be considered as covering and specifically disclosing all possible sub-ranges and individual numerical values ​​(including integers and fractions) within those ranges.

[0025] In this article, unless otherwise specified, “contains,” “includes,” “containing,” “has,” or similar terms cover the meanings of “composed of” and “mainly composed of,” for example, “A contains a” covers the meanings of “A contains a and others” and “A contains only a.”

[0026] For the sake of brevity, not all possible combinations of the technical features in each implementation scheme or embodiment are described herein. Therefore, as long as there is no contradiction in the combination of these technical features, the technical features in each implementation scheme or embodiment can be combined arbitrarily, and all possible combinations should be considered within the scope of this specification.

[0027] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0028] The following examples use instruments and equipment conventional in the art. Experimental methods in the following examples, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer. All raw materials used in the following examples are conventional commercially available products with specifications conventional in the art. In this specification and the following examples, unless otherwise specified, "%" refers to weight percentage, "parts" refers to parts by weight, and "ratio" refers to weight proportion.

[0029] Example 1 This invention discloses a method for preparing a monolithic centrifugal column (SNO-MSC) for enriching S-nitrosylated peptides, such as... Figure 1 As shown, it includes the following steps: Step 1: Preparation of monolithic spin column (MSC) First, a monolithic substrate column was prepared by in-situ thermally initiated polymerization in a polypropylene centrifuge column. The composition (mass fraction) of the prepolymer solution was as follows: 13.3% N-acryloyloxysuccinimide (NAS) as a functional monomer, 24% ethylene glycol dimethacrylate (EDMA) and 2.7% octavinyl polyhedral oligomeric silsesquioxane (OVPOS) as crosslinking agents, and 60% toluene as a porogen. After ultrasonic degassing, the mixture was fully dissolved at 70 °C, and then 1% (by mass of the monomer and crosslinking agent) of azobisisobutyronitrile (AIBN) was added as an initiator. 200 μL of the prepared prepolymer solution was added to a pre-sealed polypropylene centrifuge column, and the reaction was carried out at 70 °C for 12 h to initiate the in-situ polymerization of the monolithic material. Finally, the column was washed with methanol to remove residual reagents, yielding the monolithic substrate centrifuge column (MSC). Step 2: Pyridine dithioyl modification yields a monolithic material centrifuge column (SNO-MSC) for integrated labeling and capture of SNO. The MSCs prepared above were first swollen with acetonitrile, then equilibrated with 50 mM phosphate-buffered saline (PBS) at pH 7.4. Subsequently, 500 μL of 1 M pyridine dithioethylamine hydrochloride (PDA) was added. PBS buffer (HCl) was added to MSCs and incubated at 4 °C for 2 h. Covalent modification of pyridine disulfide groups was achieved by crosslinking the N-hydroxysuccinimide (NHS) ester group in NAS with the primary amino group in PDA. Finally, the MSCs were washed with deionized water to obtain SNO-MSCs, which were stored at 4 °C before use.

[0030] Example 2 The SNO-MSCs obtained in Example 1 were used for S-nitrosoglutathione (GSNO) extraction. First, the small molecule SNO peptide GSNO was selected as a substrate to evaluate the efficiency of SNO-MSC labeling and SNO enrichment, including the following steps: 0.2 mL of 4-hydroxyethylpiperazine ethanesulfonic acid buffer (HEPES) (100 mM, pH 7.8) containing 1 mM GSNO and 10 mM sodium ascorbate was loaded into SNO-MSCs and incubated at room temperature for 1 h. Subsequently, non-specifically adsorbed substances were removed by washing with 50% acetonitrile and HEPES buffer (50 mM, pH 7.8). Finally, GSNO adsorbed on the SNO-MSCs was eluted with 0.1 mL of ammonium bicarbonate (NH4HCO3) buffer (50 mM, pH 8.0) containing 10 mM tris(2-carboxyethyl)phosphine (TCEP) for 30 min, during which GSNO was converted to glutathione (GSH). The concentration of GSH in the eluent was determined by HPLC-MS (Agilent 1200 LC-6230 TOF MS) using an Agilent HC-C18 column (250 × 4.6 mm, 5 μm). The mobile phase consisted of 10% acetonitrile and 90% aqueous solution containing 0.1% formic acid (v / v), and eluted isocratically at a flow rate of 1 mL / min for 4 min. Mass spectrometry was performed using ESI source in positive ion mode, with the source temperature set at 300 ℃, the drying gas set at 12 L / min, the source voltage set at 3500 V, and the GSH extraction mass-to-nucleus ratio at m / z 308.0911.

[0031] To ensure the reliability and practicality of the SNO-MSCs prepared in this invention in real-world applications, GSNO was used as a model analyte to evaluate its reproducibility, reusability, and stability. The relative standard deviations (RSDs) of GSNO extraction recoveries for the same batch (n=4 columns) and different batches (n=3 batches) were 5.2% and 10.7%, respectively, indicating that SNO-MSC has good intra-batch and inter-batch reproducibility. Figure 2 As shown.

[0032] Furthermore, the SNO-MSCs developed in this application can be regenerated to their initial state after each use by adding 500 μL of a methanol solution containing 1 M diphenyl disulfide (DPD) and allowing the solution to stand at room temperature for 1 h. After each regeneration cycle, the SNO-MSCs were reused, and after 6 cycles of reuse, the GSNO extraction efficiency of the SNO-MSCs did not show a significant decrease. Figure 3 A) confirms that SNO-MSC has excellent reusability, a feature that helps to effectively reduce costs and improve resource utilization in practical applications.

[0033] The storage stability of SNO-MSCs was studied; after 5 days of storage at 4 ℃, the GSNO extraction efficiency remained almost unchanged, but after 10 days of storage, the extraction efficiency dropped to less than 80% of the initial value. Figure 3 B); Although SNO-MSC has poor long-term storage stability, its regenerable and reusable nature effectively alleviates the problem of poor long-term storage stability.

[0034] Example 3 An analytical workflow for SNO proteomics in complex biological samples was established using the SNO-MSCs obtained in Example 1: Six-week-old male C57BL / 6J mice were anesthetized by intraperitoneal injection of 0.3 mL of 1.25% tribromoethanol. Subsequently, the heart, liver, lungs, kidneys and brain tissue of each mouse were collected and immediately frozen in liquid nitrogen, and then transferred to -80°C for storage. Frozen tissue was cut into small pieces and lysed in 5 volumes of lysis buffer, which consisted of HEPES (100 mM, pH 7.8), EDTA (1 mM), copper oxychloride (0.1 mM), sodium dodecyl sulfate (SDS, 1%), Triton X-100 (1%), protease inhibitors, and PMSF. The homogenate was homogenized using a homogenizer and then sonicated on ice to extract proteins. The homogenate was centrifuged at 4 °C and 13000 × g for 20 min, and the supernatant was collected. Protein concentration was determined using a quinolinic acid (BCA) kit. The supernatant was stored at -80 °C for SNO proteomics analysis. Using mouse liver samples as a representative example, a proteomics analysis workflow for SNO in complex biological samples was established. Figure 4 ): First, the protein sample was diluted to 1 mg / mL with a buffer containing 20 mM iodoacetamide (IAA) (containing 100 mM HEPES (pH 7.8), 1 mM EDTA, and 0.1 mM new copper reagent). Then, an alkylation reaction was performed at room temperature in the dark for 40 min to block free thiol groups. After alkylation, acetone pre-cooled to -20 °C was added to precipitate the protein. The precipitate was then collected by centrifugation (12000 × g, 20 min), and impurities and excess reagents were removed. The alkylated protein precipitate was resuspended in HEPES buffer (100 mM, pH 7.8), and trypsin was added at a mass ratio of 1:40 (trypsin to protein). The mixture was incubated overnight at 37 °C to digest the protein into peptides. SNO-MSCs were swollen and equilibrated sequentially with acetonitrile and HEPES buffer (100 mM, pH 7.8). Subsequently, a peptide solution supplemented with 10 mM sodium ascorbate was added to the SNO-MSCs and incubated at room temperature for 1 h. During this period, three key processes occurred simultaneously: 1) ascorbic acid-mediated selective reduction, reducing the SNO group to a thiol group; 2) the binding of pyridine disulfide groups to the newly generated thiol groups, forming a cleavable disulfide bond; and 3) solid-phase capture of the SNO peptides using the monolithic material. Next, non-covalently bound peptides were removed by centrifugation, followed by washing twice each with 50% acetonitrile, 100 mM sodium chloride, and HEPES buffer to remove non-covalently bound peptides. Finally, the captured SNO peptides were eluted twice with NH4HCO3 (50 mM, pH 8.0) containing 10 mM TCEP for 30 min each time. The eluents were combined, concentrated and dried under vacuum. Before mass spectrometry analysis, the eluted SNO peptides were redissolved in 0.1% formic acid and desalted using an Empore™ C18 Stage tip (CDS Analytical Instruments, Pennsylvania, USA). Desalted peptides were analyzed using an Orbitrap Eclipse™ Tribrid™ mass spectrometer (Thermo Fisher Scientific, Massachusetts, USA). Chromatographic separation conditions: a reversed-phase C18 capillary column (75 μm × 20 cm, 1.9 μm) was used. The mobile phase consisted of an aqueous solution (v / v) containing 0.1% formic acid as phase A and an acetonitrile / aqueous solution (80:20, v / v) containing 0.1% formic acid as phase B. A gradient elution program was performed at a flow rate of 300 nL / min: 0–40 min, 0% → 30% phase B; 40–50 min, 30% → 50% phase B; 50–53 min, 50% → 100% phase B; 53–60 min, maintaining 100% phase B. The column temperature was set to 55 °C, and the injection volume was 1 μL. Mass spectrometry detection parameters: operation was performed in positive ion mode, with a spray voltage of 2.0 kV and a capillary temperature of 250 °C. ℃; Level 1 full scan range 350-1500 m / z, resolution 120,000, automatic gain control target value 3×10 6 Maximum injection time: 50 ms; Data-dependent mass spectrometry (DDA) mode selection: top 30 ions by intensity; isolation window: 1.4 m / z; normalized collision energy: 30%; secondary scan range: 200-2000 m / z; resolution: 15,000; automatic gain control target value: 2.5 × 10⁻⁶. 4 Maximum injection time is 20 ms, and dynamic exclusion timeout is 25 s; Database search parameters: Raw data were processed using Proteome Discoverer 2.5 software (Thermo Fisher Scientific, Massachusetts, USA) to retrieve UniProt-Mus musculus data. The hydrolase was set to trypsin, allowing a maximum of two cleavage sites to be missed; variable modifications were set to: carbamylation (C), oxidation (M), and N-terminal acetylation of protein; quality error tolerance: precursor ions 10 ppm, fragment ions 0.02 Da; the significance threshold (false discovery rate) was ≤1%.

[0035] The efficiency and selectivity of the SNO-MSCs obtained in Example 1 for integrated in-column labeling and enrichment of SNO peptides in complex biological samples were evaluated. Using mouse liver proteins as a representative sample, this study evaluated the efficiency and selectivity of SNO-MSCs for integrated intracolumn labeling and enrichment of SNO in complex biological samples containing high abundance of non-SNO protein interference. Figure 5 ); Without SNO-MSC treatment, a total of 11,138 peptides and 1,743 proteins were identified, of which only 20 peptides and 14 proteins were SNO-modified. In contrast, after SNO-MSC-based integrated labeling and enrichment of SNO within the column, a total of 8,173 peptides and 1,586 proteins were identified, of which 2,244 peptides and 939 proteins were SNO-modified. The SNO protein identification rate reached 59.2% (939 / 1586), indicating that SNO-MSC has excellent efficiency in labeling and enriching SNO peptides in complex biological matrices. To eliminate false positive results, the negative control sample (theoretically free of SNO sites after TCEP reduction) was processed using the same procedure. The results identified only four SNO peptides from three SNO proteins, with a false positive rate (FPR) of 0.3% (3 / 939). In summary, these results demonstrate that the SNO proteomics analysis workflow established in this invention exhibits highly reliable labeling and enrichment efficiency and excellent selectivity when applied to complex biological matrix samples. Therefore, this workflow has broad application prospects in large-scale SNO proteomics studies of real-world samples.

[0036] Example 4 The SNO-MSCs obtained in Example 1 were applied to the large-scale identification of SNO proteins and sites in mouse tissues: The SNO proteomics analysis workflow established in this invention was applied to the identification of endogenous SNO proteins and modification sites in different mouse tissues, with three biological replicates for each tissue sample. The results showed that 492, 1160, 907, 995, and 1029 SNO proteins were identified in the heart, liver, lung, kidney, and brain tissues of mice, respectively, and 901, 1872, 1512, 1664, and 1815 SNO modification sites were identified, respectively. Figure 6 ).

[0037] The above content is only for illustrating the technical concept of the present invention and should not be construed as limiting the scope of protection of the present invention. Any modifications made to the technical solution based on the technical concept proposed in this invention shall fall within the scope of protection of the claims of this invention.

Claims

1. A method for preparing a monolithic centrifugal column for enriching S-nitrosyl peptides, characterized in that, Includes the following steps: The monolithic material prepolymer solution was added to a pre-sealed blank polypropylene centrifuge column for thermally initiated in-situ polymerization. After the reaction was completed, the monolithic substrate centrifuge column was obtained. After swelling the monolithic substrate centrifuge column, phosphate buffer of pyridine dithioethylamine hydrochloride was added, followed by standing and washing to obtain a monolithic centrifuge column enriched with S-nitrosotriates.

2. The method for preparing a monolithic centrifuge column for enriching S-nitrosyl peptides according to claim 1, characterized in that, The prepolymer solution of the monolithic material comprises, by mass percentage, 10% to 15% N-acryloyloxysuccinimide, 20% to 30% ethylene glycol dimethacrylate, 1% to 3% octavinyl polyhedral oligomeric silsesquioxane, 55% to 65% toluene and azobisisobutyronitrile; The content of the azobisisobutyronitrile is 1% to 2% of the total mass of N-acryloyloxysuccinimide and octavinyl polyhedral oligomeric silsesquioxane.

3. The method for preparing a monolithic centrifuge column for enriching S-nitrosylated peptides according to claim 1, characterized in that, The method for preparing the prepolymer solution of the monolithic material is as follows: N-acryloyloxysuccinimide, ethylene glycol dimethacrylate, octavinyl polyhedral oligomeric silsesquioxane and toluene were mixed, degassed by ultrasound, dissolved at 65-75 °C, and then azobisisobutyronitrile was added. After mixing, a prepolymer solution of the whole material was obtained.

4. The method for preparing a monolithic centrifuge column for enriching S-nitrosylated peptides according to claim 1, characterized in that, Add 150-300 μL of prepolymer solution of the whole material to an 800 μL polypropylene centrifuge column.

5. The method for preparing a monolithic centrifuge column for enriching S-nitrosylated peptides according to claim 1, characterized in that, The temperature for the thermally initiated in-situ polymerization reaction is 65~75 ℃, and the reaction time is 12~24 h.

6. The method for preparing a monolithic centrifuge column for enriching S-nitrosyl peptides according to claim 1, characterized in that, After the substrate monolithic material centrifuge column was swollen with acetonitrile, it was equilibrated with phosphate buffer. Then, phosphate buffer of pyridine dithioethylamine hydrochloride was added to the substrate monolithic material. The concentration of the phosphate buffer was 50~100 mM and the pH value was 7.4~8.

2.

7. A method for preparing a monolithic centrifuge column for enriching S-nitrosylated peptides according to claim 1, characterized in that, In the phosphate buffer solution of pyridine dithioethylamine hydrochloride, the concentration of pyridine dithioethylamine hydrochloride is 0.5~2M; the volume of the phosphate buffer solution of pyridine dithioethylamine hydrochloride is 200~800 μL. The placement temperature is 2~6 ℃, and the time is 2~4 h.

8. A monolithic centrifugal column for enriching S-nitrosylated peptides, characterized in that, It is prepared by the preparation method described in any one of claims 1 to 7.

9. The integral centrifuge column for enriching S-nitrosylated peptides as described in claim 8 is used for the integrated reduction, labeling, enrichment of S-nitrosylated peptides and its application in S-nitrosyl proteomics analysis of complex biological samples; Its features are, The specific steps for S-nitroso proteomics analysis are as follows: After alkylating the protein sample dilution solution, pre-cooled acetone was added to precipitate the protein. The protein precipitate was then collected by centrifugation to obtain the alkylated protein precipitate. The alkylated protein precipitate was resuspended in HEPES buffer, and then incubated with trypsin to obtain the enzymatically digested peptide solution. Sodium ascorbate was added to the peptide solution, and then added to a monolithic centrifuge column enriched with S-nitrosyl peptides that had been swollen and equilibrated. The column was then placed at room temperature to perform the integrated reduction, labeling, and enrichment of S-nitrosyl peptides, resulting in a monolithic centrifuge column enriched with S-nitrosyl peptide samples. Elution solution was added to the whole centrifuge column enriched with S-nitrosyl peptides, and the column was placed at room temperature to elute the S-nitrosyl peptides, thus obtaining the eluted peptide sample. The eluted peptide samples were desalted and analyzed by liquid chromatography-mass spectrometry to identify S-nitroso proteins and modification sites. The protein sample dilution buffer is prepared by diluting the protein sample to 0.5-2 mg / mL with HEPES buffer containing 10-40 mM iodoacetamide; the concentration of the HEPES buffer is 50-100 mM and the pH value is 7.2-7.8; the alkylation reaction is carried out at room temperature and in the dark for 30-60 min. The temperature of the pre-cooled acetone is -15 to -20°C; the volume ratio of acetone to protein sample dilution is (3 to 5):

1. The mass ratio of trypsin to protein precipitate is 1:20 to 1:50; The final concentration of sodium ascorbate is 10-20 mM; the integrated reduction, labeling, and enrichment of S-nitrosotropic peptides are carried out by placing the product at room temperature for 1-2 h. The elution solution is a TCEP or DTT solution with a concentration of 5-20 mM, and the elution time is 0.5-2 h.

10. The monolithic centrifuge column for enriching S-nitrosylated peptides according to claim 9, and its application in the integrated reduction, labeling, enrichment, and S-nitrosylated peptide proteomics analysis of complex biological samples, is characterized in that... After the integrated reduction, labeling, and enrichment of S-nitrosolated peptides, the monolithic centrifugal column for enriching S-nitrosolated peptides is regenerated by adding 200-800 μL of methanol solution containing 0.5-2 M diphenyl disulfide and leaving it at room temperature for 1-5 h to restore it to its initial state for reuse. The number of times the device is reused is no less than 6.

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