Method for simultaneously detecting protein phosphorylation and S-nitrosation

Abstract

The invention discloses a method for simultaneously detecting protein phosphorylation and S-nitrosation, which comprises the following steps of: marking S-nitrosation protein by using a phosphoric acid affinity label, and converting S-nitrosation modification into a phosphorylation simulation type label; therefore, the conventional phosphorylated proteomics technology can be used for simultaneously enriching and analyzing two modifications of S-nitrosation and phosphorylation, and tests on model proteins and complex biological samples prove that the method is high in sensitivity and good in repeatability, has a wide protein coverage range, is equivalent to a traditional independent enrichment method, and has a wide application prospect. The technical challenge of analyzing modification of different chemical properties is overcome, and a reliable tool is provided for comprehensively researching PTM cross dialogue.

Description

Technical Field

[0001] The present invention relates to the technical field of proteomics methods, and particularly relates to a method for simultaneously detecting protein phosphorylation and S-nitrosylation. Background Art

[0002] Post-translational modifications (PTMs) are dynamic regulators of protein function and activity. Among the more than 400 known post-translational modifications (PTMs), reversible phosphorylation has become the most intensively studied PTM type due to its central role in eukaryotic signal transduction, protein-protein interaction, enzyme activity regulation, and subcellular localization. At the same time, protein S S-nitrosylation (a modification method that links nitric oxide (NO) molecules to cysteine residues of proteins through redox reactions) has also attracted much attention due to its regulatory function similar to phosphorylation. Recent studies have revealed that S-nitrosylation is widely involved in the molecular mechanisms of plant growth and development regulation and stress response.

[0003] Proteins usually undergo different types of modifications at multiple sites, and the interaction between different post-translational modifications (PTMs) adds an additional level of regulation to the proteome and makes it more complex. More and more studies have shown that there is a complex crosstalk between phosphorylation and S-nitrosylation. For example, in Arabidopsis thaliana, the phosphorylation of salicylic acid-binding protein SABP3 requires prior S-nitrosylation, while in the BIK1 protein, phosphorylation occurs before S-nitrosylation, jointly regulating immune signals. This interaction is not limited to individual proteins, and PTMs of certain kinases can also affect the entire signal pathway by changing the phosphorylation state of downstream substrates. Particularly noteworthy is that S-nitrosylation can regulate the activity of multiple kinases, including sucrose non-fermenting-1-related protein kinase (SnRK2), target of rapamycin (TOR), FERONIA receptor kinase (FER), mitogen-activated protein kinase (MAPKs), and thioredoxin-interacting receptor kinase (TIRK). This indicates that the complex crosstalk between S-nitrosylation and phosphorylation plays an important regulatory role in plant cell signal transduction.

[0004] Although scientists are increasingly interested in the interaction between S-nitrosylation and phosphorylation, simultaneously analyzing S-nitrosylation and phosphorylation faces technical difficulties. The abundances of these two modifications in cells are very low, so selective enrichment is a key step for effective detection using mass spectrometry (MS). However, the properties of S-nitrosylation and phosphorylation modifications are very different, and different enrichment strategies are required: phosphorylated proteins / peptides are usually directly enriched for phosphorylated groups using metal oxide / ion affinity chromatography (MOAC / IMAC), while S-nitrosylated proteins / peptides rely on tag conversion strategies (converting S-nitrosylation modifications into other enrichable groups), such as the traditional biotin switch technique (BST) that converts S-nitrosylation into biotin groups. Scientists have successively developed a series of new tag conversion techniques, such as isotope-coded affinity tags (ICAT) (Wu CG, Parrott AM, Liu T, et al. Distinction of thioredoxin transnitrosylation and denitrosylation target proteins by the ICAT quantitative approach. J. Proteomics 2011, 74(11): 2498-2509.), bioorthogonal cleavable Cys-BOOST (Mnatsakanyan R, Markoutsa S, Walbrunn K, et al. Proteome-wide detection of S-nitrosylation targets and motifs using bioorthogonal cleavable-linker-based enrichment and switch technique. Nat. Commun. 2019, 10(1): 2195), iodo-TMT of iodoacetylated tandem mass tags (Gong B, Shi QH. Identifying S-nitrosylated proteins and unraveling S-nitrosoglutathione reductase-modulated sodic alkaline stress tolerance in Solanum lycopersicum L. Plant Physiol. Biochem. 2019, 142: 84-93.), and fluorine affinity tags (FAT) (Qin GC, Qu MH, Jia B, et al.FAT-switch-based quantitative S-nitrosoproteomics reveals a key role of GSNOR1 in regulating ER functions. Nat. Commun. 2023, 14(1): 3268.). However, these conversion tags for S-nitrosylation modification are different in nature from those of phosphorylation modification, resulting in the inability to enrich both simultaneously. If one wants to analyze these two modifications simultaneously, enrichment and analysis need to be carried out separately or sequentially, which is not only impractical for studies with limited sample sizes but also brings a series of problems, such as differences in enrichment efficiency, increased experimental complexity and cost, reduced throughput, and sample loss due to non-specific adsorption. Therefore, the simultaneous analysis of protein phosphorylation and S-nitrosylation modification still faces technical bottlenecks. Summary of the Invention

[0005] The object of the present invention is to provide a method for simultaneously detecting protein phosphorylation and S-nitrosylation, which solves the problem that the prior art cannot enrich and detect phosphorylation and S-nitrosylation simultaneously.

[0006] The present invention is achieved by the following technical solutions: A method for simultaneously detecting protein phosphorylation and S-nitrosylation (SNO), the method comprising the following steps: 1) Label S-nitrosylated proteins with a phosphorylated affinity tag (PAT) to convert the S-nitrosylation modification into a phosphorylation-mimicking tag (this method is named PAT-switch); the phosphorylated affinity tag (PAT) consists of three key parts: a thiol-reactive iodoacetyl group, an alkyl spacer, and a specific phosphate tag; 2) Use conventional phosphoproteomics techniques to simultaneously enrich and analyze S-nitrosylated peptides (already PAT-switched) and phosphorylated peptide segments in the digest of the proteins labeled in step 1), and the conventional phosphoproteomics techniques include simultaneously enriching S-nitrosylated peptides (already PAT-switched) and phosphorylated peptides through metal oxide / immobilized metal ion affinity chromatography (MOAC / IMAC), and then jointly detecting them by liquid chromatography-tandem mass spectrometry (LC-MS / MS).

[0007] Preferably, the phosphorylated affinity tag in step 1) is iodoacetamido-LC-phosphonic acid (6C-CysPAT), and its structure is: , because it is not only easily accessible, but also its thiol reactivity of iodoacetyl group has been fully verified. In addition, through pretreatment strategies such as Cys-BOOST or FAT, specific labeling of S-nitrosylation modification can also be achieved. These strategies include blocking free thiols, reducing and labeling S-nitrosylation modification, and reducing disulfide bonds.

[0008] Specifically, before proteolytic digestion of the labeled protein, it also includes reduction treatment and alkylation treatment of disulfide bonds, and the reagents used include tris(2-carboxyethyl)phosphine / 2-chloroacetamide (TCEP / CAA).

[0009] S-nitrosylation modification occurs on the thiol group of cysteine in proteins, and cysteine mainly exists in three forms in proteins: free thiol, S-nitrosylated cysteine, and disulfide bond. Due to their different chemical properties, they need to be treated separately. First, free thiols need to be blocked with iodoacetamide (IAA) before protein labeling, retaining S-nitrosylated cysteine and disulfide bonds; then impurities are removed by protein precipitation, and then S-nitrosylation modification is reduced with sodium ascorbate, and labeling is achieved with 6C-CysPAT (S-nitrosylated cysteine is converted to PAT-labeled cysteine). Subsequently, protein is precipitated with acetone to remove excess 6C-CysPAT reagent; after redissolving the protein, disulfide bonds are reduced and blocked with TCEP / CAA. Finally, the protein is digested into peptides by trypsin to obtain a mixture of phosphorylated peptides, PAT-labeled peptides, and unmodified peptides; phosphorylated peptides and PAT-labeled peptides are simultaneously enriched by metal oxide / immobilized metal ion affinity chromatography technology, while unmodified peptides are eluted; the enriched peptides are analyzed by liquid chromatography-tandem mass spectrometry (LC-MS / MS). Due to the different mass shifts of phosphorylation and PAT labeling (phosphorylation is 79.97 Da, and the PAT tag is 221.08 Da), the mass spectrometer can clearly distinguish these two modifications, and cooperate with tandem mass spectrometry to achieve identification of polypeptide sequences and modification sites, as well as quantitative analysis.

[0010] Before protein labeling, free thiols are blocked with iodoacetamide (IAA), and then impurities are removed by protein precipitation. The specific steps are as follows: The protein is resuspended in HENS buffer, iodoacetamide (IAA) is added to a final concentration of 100 mM, and then incubated with shaking (600 rpm) at 37°C for 1 hour. The mixture is vortexed, acetone is added to precipitate the protein, or the protein is extracted with phenol and then methanol is added to precipitate the protein. After centrifugation, the protein precipitate is washed and dried in air.

[0011] Reduce the S-nitrosylation modification with sodium ascorbate and label it with 6C-CysPAT. Subsequently, precipitate the protein with acetone to remove the excess 6C-CysPAT reagent. The specific steps are as follows: Resuspend the protein precipitate in HENS buffer containing sodium ascorbate and 6C-CysPAT, gently roll and incubate at 37 °C for 1 hour, and then perform rapid protein precipitation (mix the mixture with 1 / 10 volume of 1 M NaCl and co-incubate with 5 volumes of acetone at room temperature for 30 minutes). After centrifugation, wash the protein precipitate twice with 80 vol% acetone (containing 10 mM NaCl) and dry it in air for 5 minutes to obtain the protein with S-nitrosylation modification switched by PAT.

[0012] Reduce and block the disulfide bonds with TCEP / CAA. Finally, the protein is digested into peptides by trypsin to obtain a mixture of phosphorylated peptides, PAT-labeled peptides, and unmodified peptides. The specific steps are as follows: Resuspend the protein precipitate after PAT-Switch labeling in denaturing buffer containing TCEP / CAA and incubate at 60 °C for 30 minutes. Sequentially add 4 volumes of methanol, 1 volume of chloroform, and 3 volumes of water of the denaturing buffer. After centrifugation, wash the precipitate with cold methanol (containing 0.1 M NH 4 OAc) and 80 vol% acetone (containing 10 mM NaCl) and dry it in air for 5 minutes; then resuspend it in 50 mM ammonium bicarbonate buffer, add trypsin (1 / 50 of the protein mass), and incubate at 37 °C for 12 hours. Add formic acid to the digestion solution to acidify it to 1 vol%, desalt it using a peptide desalting centrifugal column, quantify it using the Pierce Quantitative Colorimetric Peptide Assay Kit, and dry it under vacuum to obtain.

[0013] The enriched peptides were analyzed by liquid chromatography - tandem mass spectrometry under the following conditions: separation was carried out on a C18 analytical column using an acetonitrile gradient (5% - 25%, 0 - 90 minutes; 25% - 35%, 90 - 105 minutes; 35% - 85%, 105 - 120 minutes) at a flow rate of 300 nL / min; the source voltage was set to 2.0 kV and the ion transfer tube temperature was maintained at 320 °C; the mass spectrometer was operated in data - dependent acquisition (DDA) and data - independent acquisition (DIA) modes; in the DDA mode, MS1 scans were performed by an Orbitrap detector using 400−1300 m / z, 120 K resolution, 60% RF lens, 8E5 AGC target, and a maximum ion injection time of 1100 ms, while MS2 scans were carried out with a 5.0E4 intensity threshold, 2 - 6 charge states, 25 s exclusion time, 54 ms maximum ion injection time, 1.6 Da isolation window, 30% HCD collision energy, 1.0E5 AGC target, and a 3 s cycle time; background ions with m / z = 519.1388 (C 2 H 6 SiO) 7 H + were used for internal calibration.

[0014] Preferably, to enhance peptide identification and data - independent acquisition (DIA) quantification, before enrichment and analysis, we fractionated the mixed peptides using high - pH reversed - phase high - performance liquid chromatography (RP - HPLC). A Waters XBridgeBEH130 C18 column (3.5 μm, 4.6 × 250 mm) and an Ultimate 3000 HPLC system (Dionex, California, USA) were used to fractionate 3.6 mg of peptides at a flow rate of 1 mL / min. The separation was carried out using buffer A (10 mM ammonium formate, pH 10) and buffer B (10 mM ammonium formate, containing 90% acetonitrile, pH 10) with a gradient of 60 minutes: 1% to 25% buffer B in 48 minutes, increasing to 60% in 5 minutes, increasing to 70% in 2 minutes, maintaining 70% for 5 minutes, and then returning to 1%. UV absorbance was monitored at 214 and 280 nm. Fractions were collected every minute for the first 60 minutes, and 60 fractions were equally spaced and combined into 12 fraction groups (e.g., fractions 1, 13, 25, 37, 49 were in one group), and then vacuum - dried.

[0015] The beneficial effects of the present invention are as follows: 1) The present invention uses a phosphoric acid affinity tag (PAT) to label S-nitrosylated proteins, converting the S-nitrosylation modification into a phosphorylation-mimicking tag, so that conventional phosphoproteomics techniques can be used to simultaneously enrich and analyze these two modifications, namely S-nitrosylation and phosphorylation. Through the testing of model proteins and complex biological samples, we have verified that this method has high sensitivity, good repeatability, a wide protein coverage range, is comparable to traditional single enrichment methods, overcomes the technical challenges of analyzing modifications with different chemical properties, and provides a reliable tool for comprehensively studying PTM cross-talk.

[0016] 2) By obtaining dual omics datasets in the same workflow, the present invention significantly improves the repeatability and cost-effectiveness of experiments. Compared with parallel or sequential single omics analysis, this method reduces manpower, reagent consumption, and instrument usage time. At the same time, the consistency of the two omics datasets provides a more reliable basis for biological research.

[0017] 3) More importantly, this method provides a powerful tool for studying plant signal transduction, stress response, and metabolic regulation. Based on this, we have discovered many new S-nitrosylation sites and revealed potential PTM interactions in key regulatory proteins. Brief Description of the Drawings

[0018] Figure 1 is a schematic diagram of the technological process of the present invention; Figure 2 is a diagram showing the enrichment ability of a phosphoric acid column for S-nitrosylated BSA digested polypeptides in Example 1, where A is the mass spectrometry diagram of 3 samples in the untreated group; B is the mass spectrometry diagram of 2 samples without enrichment; C is the mass spectrometry diagram of the samples in Group 1; D is a diagram for sensitivity evaluation of trace S-nitrosylated BSA (100 fmol).

[0019] Figure 3 is a diagram for verifying the feasibility of analyzing protein phosphorylation and S-nitrosylation in biological samples in Example 2; where A is a comparison of polypeptide coverage with single omics technology; B is the number of phosphorylated peptide identifications before and after PAT treatment.

[0020] Figure 4 is a diagram for evaluating the reproducibility of analyzing protein phosphorylation and S-nitrosylation in biological samples in Example 2; where A is a comparison of total ion current (TIC) diagrams of three parallel experiments; B and C are scatter plots of polypeptide abundances between technical replicates in data-dependent acquisition (DDA) and data-independent acquisition (DIA) modes respectively; D is the correlation between biological replicates.

[0021] Figure 5This is the statistical analysis for large-scale identification of phosphorylation and S-nitrosylation sites in Arabidopsis thaliana in Example 3, where A represents the total number of identified modified polypeptides and sites; B represents the number of identified phosphorylated, S-nitrosylated proteins, and doubly modified proteins. Detailed implementation mode

[0022] The following is a further description of the present invention, rather than a limitation thereof.

[0023] Reagents and materials: Bovine serum albumin (BSA), iodoacetamide (IAA), 2-chloroacetamide (CAA), dithiothreitol (DTT), tris(2-carboxyethyl)phosphine (TCEP), acetone, and chloroform used in the experiment were all purchased from Sigma-Aldrich (Missouri, USA). LC-MS grade acetonitrile (ACN), water, methanol, formic acid (FA), iodoacetamide-LC-phosphoric acid (6C-CysPAT), protease and phosphatase inhibitor mini tablets, bicinchoninic acid (BCA) protein assay kit, Zeba protein desalting centrifugal column, and Pierce™ peptide desalting centrifugal column were from Thermo Fisher Scientific (Massachusetts, USA). HEPES, EDTA, neocuproine, sucrose, sodium dodecyl sulfate (SDS), ammonium acetate (NH 4 OAc), sodium chloride (NaCl), sodium nitrite (NaNO 2 ), hydrochloric acid (HCL), sodium ascorbate, urea, and ammonium bicarbonate (NH 4 HCO 3 ) were purchased from Aladdin Science (Shanghai, China). Sequencing grade trypsin was provided by Promega Corporation (Wisconsin, USA).

[0024] The mixing grinder (MM400) and ultrasonic disruptor (Bioruptor UCD-300) were purchased from RETSCH (Germany) and Diagenode (Belgium), respectively. The centrifuge (Multifuge 1S-R), nano liquid chromatography (Ultimate™ 3000RSLCnano), and mass spectrometer (Orbitrap Fusion) were from Thermo Fisher Scientific (San Jose, California, USA).

[0025] Example 1: Verification of enrichment effect using a model protein To evaluate its enrichment ability for PAT-labeled peptides, we used artificially S-nitrosylated bovine serum albumin (S-nitrosylated BSA) as a model protein.

[0026] Among them, the preparation of artificial S-nitrosylated bovine serum albumin (S-nitrosylated BSA): According to the method of Wang YA et al. (Wang YA, Liu XH, Li KY, et al. Self-Sulfhydrated, Nitro-Fixed Albumin Nanoparticles as a Potent Therapeutic Agent for the Treatment of Acute Liver Injury. ACS Nano 2024, 18(31): 20772-20791.), bovine serum albumin (BSA) was first reduced and then S-nitrosylated with sodium nitrite to prepare artificial S-nitrosylated BSA (S-nitrosylated BSA). Briefly, the steps are as follows. Bovine serum albumin (BSA) was dissolved in 0.1 M Tris-Cl buffer (pH 8.5) at a concentration of 5 mg·mL –1 , and then reduced with 20 mM TCEP at 37 °C for 30 minutes. After two rounds of desalting with Zeba spin columns, the pH was adjusted to 1.0 with HCl, and the solution was mixed with an equal volume of NaNO 2 (about 20-fold molar excess relative to BSA thiols). The mixture was incubated in the dark at 25 °C for 1 hour. Finally, it was precipitated with 4 volumes of cold acetone at -20 °C for 2 hours and washed twice with cold acetone and methanol.

[0027] The prepared S-nitrosylated BSA samples were divided into three groups, denoted as Group 1 - Group 3. Among them, the samples in Group 1 were processed as follows: Free thiols were blocked with iodoacetamide (IAA), PAT-Switch labeling was performed, and after labeling, the protein was reduced, alkylated, and enzymatically digested and enriched; the samples in Group 2 were processed with reference to the samples in Group 1, except that enrichment was not performed; the samples in Group 3 were processed with reference to the samples in Group 1, except that PAT-Switch labeling was not performed and enrichment was not performed, serving as a blank control. After treatment, the protein was enzymatically digested into peptide segments and then analyzed by liquid chromatography-tandem mass spectrometry.

[0028] Among them, the specific process of processing the samples in Group 1 is as follows: 1) Block free thiols in S-nitrosylated bovine serum albumin (S-nitrosylated BSA) with iodoacetamide (IAA): Resuspend 1 mg of artificial S-nitrosylated bovine serum albumin (S-nitrosylated BSA) in 500 μL of HENS buffer (100 mM HEPES, pH 7.7, 1 mM EDTA, 0.1 mM neocuproine, 1 wt% SDS), add iodoacetamide (IAA) to a final concentration of 100 mM, then incubate with shaking (600 rpm) at 37°C for 1 hour. Mix the mixture with 50 μL of 1 M NaCl and incubate with 2.5 mL of acetone at room temperature for 30 minutes. After centrifugation (8,000 × g, 10 minutes, 4°C), wash the protein precipitate twice with 80 vol% acetone (containing 10 mM NaCl) and dry it in air for 5 minutes.

[0029] 2) Reduce S-nitrosylation modification and PAT-Switch labeling: All operations are carried out under low light conditions. Resuspend the protein precipitate obtained in step 1) in 500 μL of HENS buffer (containing 20 mM sodium ascorbate and 5 mM 6C-CysPAT). Shake the mixture at 37°C for 1 hour, then perform rapid protein precipitation (mix the mixture with 50 μL of 1 M NaCl and incubate with 2.5 mL of acetone at room temperature for 30 minutes). After centrifugation (8,000 × g, 10 minutes, 4°C), wash the protein precipitate twice with 80 vol% acetone (containing 10 mM NaCl) and dry it in air for 5 minutes to obtain S-nitrosylated protein after PAT-Switch labeling. (Skip this step for the untreated group 3 sample control).

[0030] 3) Reduce, alkylate, and digest S-nitrosylated protein after PAT-Switch labeling: Resuspend the protein precipitate obtained in step 2) (S-nitrosylated protein after PAT-Switch labeling) in 500 μL of denaturing buffer (8 M urea, 50 mM ammonium bicarbonate, 10 mM TCEP, and 40 mM CAA) and incubate at 60°C for 30 minutes. Perform precipitation using the chloroform-methanol method, adding 2 mL of methanol, 0.5 mL of chloroform, and 1.5 mL of water in sequence. After centrifugation (8,000 × g, 10 minutes, 4°C), wash the precipitate with cold methanol (containing 0.1 M NH 4washed with OAc) and 80 vol% acetone (containing 10 mM NaCl), and dried in air for 5 minutes. The precipitate was resuspended in 500 μL of 50 mM ammonium bicarbonate buffer, trypsin (1 / 50 of the protein mass) was added, and incubated at 37 °C for 12 hours. Formic acid was added to the digestion solution to acidify to 1 vol%, desalted using a peptide desalting centrifugal column, quantified using the Pierce Quantitative Colorimetric Peptide Assay Kit, and dried under vacuum.

[0031] 4) Enrichment and LC-MS / MS analysis: The peptide mixture obtained in step 3) was resuspended in loading buffer (80 vol% ACN, 3 vol% TFA) and loaded onto a phosphoric acid capture column for online enrichment. After washing away non-specifically bound peptides with the loading buffer, the target peptides were eluted with 1 M NH 4 H 2 PO 4 and subsequently captured on a downstream C18 capture column (samples in groups 2-3 do not need to be enriched with a phosphoric acid capture column and are directly loaded onto the C18 capture column). The enriched peptides were separated on a C18 analytical column (75 μm inner diameter × 250 mm, 1.9 μm) (Omicsolution Co., Ltd) using an acetonitrile gradient (5%-35%, 0-10 minutes) at a flow rate of 300 nL / min. The source voltage was set to 2.0 kV, and the ion transfer tube temperature was maintained at 320 °C. The mass spectrometer was operated in data-dependent acquisition (DDA) and data-independent acquisition (DIA) modes respectively. In the DDA mode, the MS1 scan was performed by the Orbitrap detector using 400−1300 m / z, 120 K resolution, 60% RF lens, 8E5 AGC target, and 1100 ms maximum ion injection time, while the MS2 scan used a 5.0E4 intensity threshold, 2-6 charge states, 25 s exclusion time, 54 ms maximum ion injection time, 1.6 Da isolation window, 30% HCD collision energy, 1.0E5 AGC target, and 3 s cycle time. Background ions (C 2 H 6 SiO) 7 H + with m / z = 519.1388 were used for internal calibration (lock mass).

[0032] 5) Database search: Proteome Discoverer™ 3.1 software and the SEQUEST search engine were used for peptide identification and label-free quantification (LFQ). The original file was searched against the BSA FASTA file (P02769 in Swiss-Prot). Trypsin was selected as the enzyme, and a maximum of two missed cleavage sites were allowed. The precursor mass tolerance was set to 10ppm, and the fragment mass tolerance was set to 0.02 Da. Variable modifications included 6C-CyaPAT (C), oxidation (M), and carbamidomethylation (C), and a maximum of five modifications were allowed for each peptide. The false discovery rate (FDR) of peptides was calculated using the Percolator node algorithm, with high and medium confidence thresholds of 0.01 and 0.05, respectively.

[0033] Mass spectrometry analysis showed that in the non-enriched S-nitrosylated BSA enzymatic hydrolysate, the signal of the unmodified peptide was very strong, while the signal of the S-nitrosylated peptide was very weak ( Figure 2 PAT labeling alone without enrichment did not significantly improve the detection of S-nitrosylated peptides ( Figure 2 However, PAT labeling and post-enrichment analysis of group 1 samples significantly enhanced the signal of PAT-labeled peptides ( Figure 2 C in ). We identified 30 S-nitrosylated sites among a total of 35 cysteine ​​residues in BSA. Although the initial abundance of unmodified peptides was 1000-fold higher, they were effectively removed after enrichment. Sensitivity analysis of the enrichment and analysis pipeline using trace amounts of S-nitrosylated BSA (100 fmol) resulted in the detection of seven PAT-tagged peptides ( Figure 2 D), demonstrating the high specificity and sensitivity of this method.

[0034] Example 2: Testing method performance in complex biological samples After verifying the effectiveness of the method of the present invention (PAT-switch method) for S-nitrosylated BSA in Example 1, we further tested its ability to simultaneously analyze phosphorylation and S-nitrosylation in complex biological samples. We compared this method with two cutting-edge technologies - FAT [Qin GC, Qu MH, Jia B, et al. FAT-switch-based quantitative S-nitrosoproteomics reveals a key role of GSNOR1 in regulating ER functions. Nat. Commun. 2023, 14(1): 3268.] and GreenPhos [Duan XX, ZhangYY, Huang XH, et al. GreenPhos, a universal method for in-depth measurement of plant phosphoproteomes with high quantitative reproducibility. Mol. Plant 2024, 17(1): 199-213.], the former for S-nitrosoproteomics and the latter for phosphoproteomics. We used Arabidopsis thaliana seedling samples, which were processed by the PAT-switch method and then subjected to three single injections for online enrichment and LC-MS / MS analysis.

[0035] The specific steps are as follows: 1) Total protein extraction from Arabidopsis thaliana and treatment for blocking free thiols: All operations were carried out under low temperature and weak light conditions, and metal-free reagents were used (unless otherwise specified). First, Arabidopsis thaliana seedlings were ground into fine powder in liquid nitrogen using a mixer mill. Approximately 0.2 g of the powder was resuspended in 2 mL of cold HENS buffer (100 mM HEPES, pH 7.7, 1 mM EDTA, 0.1 mM neocuproine, 1 wt% SDS), which also contained 0.9 M sucrose and was supplemented with protease / phosphatase inhibitors. Proteins were extracted by 10 cycles of sonication (10 s on / 10 s off) in an ice bath. The homogenate was centrifuged at 16,000 × g for 15 minutes at 4°C, and the supernatant was collected. Iodoacetamide (IAA) was added to a final concentration of 100 mM, and then the mixture was incubated with shaking (600 rpm) at 37°C for 1 hour. The solution was mixed with an equal volume of Tris-saturated phenol (pH 8.0), vortexed for 10 minutes, and centrifuged at 8,000 × g for 20 minutes at 4°C. The upper phenol phase was collected and mixed with 5 volumes of pre-cooled methanol (containing 0.1 M NH4 Mix with OAc and incubate at -20 °C for 3 hours. Precipitate the protein by centrifugation (8,000 × g, 20 minutes, 4 °C), and wash with cold methanol (containing 0.1 M NH 4 OAc) and 80 vol% acetone (containing 10 mM NaCl), and air-dry for 5 minutes.

[0036] 2) Reduction of S-nitrosylation modification and PAT labeling: Resuspend the protein precipitate obtained in step 1) in 500 μL of HENS buffer, which contains 20 mM sodium ascorbate and 5 mM 6C-CysPAT. Incubate the mixture with shaking at 37 °C for 1 hour. Vortex the mixture with 50 μL of 1 M NaCl and incubate with 2.5 mL of acetone at room temperature for 30 minutes. After centrifugation (8,000 × g, 10 minutes, 4 °C), wash the protein precipitate twice with 80 vol% acetone (containing 10 mM NaCl) and air-dry for 5 minutes.

[0037] 3) Protein reduction, alkylation, and digestion after PAT-Switch labeling: Resuspend the protein precipitate obtained in step 2) in denaturing buffer (8 M urea, 50 mM ammonium bicarbonate, 10 mM TCEP, and 40 mM CAA) and incubate at 60 °C for 30 minutes. Sequentially add 4 volumes of methanol, 1 volume of chloroform, and 3 volumes of water to the denaturing buffer. After centrifugation (8,000 × g, 10 minutes, 4 °C), wash the precipitate with cold methanol (0.1 M NH 4 OAc) and 80 vol% acetone (containing 10 mM NaCl) and air-dry for 5 minutes. Resuspend the precipitate in 500 μL of 50 mM ammonium bicarbonate buffer, add trypsin (1 / 50 of the protein mass), and incubate at 37 °C for 12 hours. Acidify the digestion solution to 1 vol% with formic acid, desalt using a peptide desalting centrifugal column, quantify using the Pierce Quantitative Colorimetric Peptide Assay Kit, and vacuum-dry.

[0038] 4) Enrichment and LC-MS / MS analysis: Resuspend the peptide mixture obtained in step 3) in loading buffer (80 vol% ACN, 3 vol% TFA) and load it onto a phosphoric acid capture column for online enrichment. After washing away non-specifically bound peptides with the loading buffer, use 1 M NH 4 H 2 PO 4Elute the target peptide and subsequently capture it on the downstream C18 capture column. The enriched peptides are separated on a C18 analytical column (75 μm inner diameter × 250 mm, 1.9 μm) (Omicsolution Co., Ltd) using an acetonitrile gradient (5% - 25%, 0 - 90 minutes; 25% - 35%, 90 - 105 minutes; 35% - 85%, 105 - 120 minutes) at a flow rate of 300 nL / min. The source voltage is set to 2.0 kV, and the ion transfer tube temperature is maintained at 320 °C. The mass spectrometer operates in data-dependent acquisition (DDA) and data-independent acquisition (DIA) modes. In DDA mode, the MS1 scan is performed by the Orbitrap detector using 400−1300 m / z, 120 K resolution, 60% RF lens, 8E5 AGC target, and a maximum ion injection time of 1100 ms, while the MS2 scan uses a 5.0E4 intensity threshold, 2 - 6 charge states, 25 s exclusion time, 54 ms maximum ion injection time, 1.6 Da isolation window, 30% HCD collision energy, 1.0E5 AGC target, and a 3 s cycle time. Use the background ion (C 2 H 6 SiO) 7 H + for internal calibration (lock mass).

[0039] 5) Database search: Peptide identification and label-free quantification (LFQ) are performed using Proteome Discoverer™ 3.1 software and the SEQUEST search engine. The raw files are searched against the FASTA file of the Arabidopsis thaliana proteome (TAIR10, EnsemblPlants). Trypsin is selected as the enzyme, allowing a maximum of two missed cleavage sites. The precursor mass tolerance is set to 10 ppm, and the fragment mass tolerance is set to 0.02 Da. Variable modifications include 6C-CyaPAT(C), phosphorylation (S, T, Y), oxidation (M), and carbamidomethylation (C), allowing a maximum of five modifications per peptide. The false discovery rate (FDR) of peptides is calculated using the Percolator node algorithm, and the thresholds for high-confidence and medium-confidence are 0.01 and 0.05, respectively. The phosphorylation and S-nitrosylation probabilities at each site are evaluated using ptmRS, with a p-value cutoff of 0.75, and sites above this are class I confidence sites.

[0040] This method identified 2,442 S-nitrosylated peptides and 4,959 phosphorylated peptides from Arabidopsis seedlings (A in Figure 3). When using 300 μg of protein, the coverage of phosphorylated peptides was slightly lower than that of GreenPhos (5,703), while the S-nitrosylation performance was comparable to that of FAT (2,559). 4,201 phosphorylated peptides were identified in the sample without PAT treatment (i.e., conventional phosphoproteomic analysis), while 4,465 phosphorylated peptides were identified in the sample treated with PAT ( Figure 3 B in), indicating that PAT labeling has no interference with phosphorylation analysis. In addition, increasing the LC-MS / MS gradient time from 120 minutes to 240 minutes only led to a slight increase in the number of phosphorylated peptides identified ( Figure 3 B in), indicating that a 120-minute gradient is an appropriate analysis condition.

[0041] The quantitative reproducibility was evaluated by repeated analysis, and the base peak chromatograms in three identical runs were similar ( Figure 4 A in). For parallel enrichment and LC-MS / MS analysis, the Pearson correlation coefficients of the intensities of modified peptides between repeats were high, reaching 0.982 and 0.973 in DDA and DIA modes, respectively (B in Figure 4, Figure 4 C in). Biological replicates across the entire workflow showed excellent reproducibility, with a Pearson correlation coefficient of 0.967 ( Figure 4 D in). In contrast, the correlation coefficients reported by GreenPhos were 0.97 and 0.95, respectively (data from the literature), indicating a slight improvement in the reproducibility of the new method.

[0042] Example 3: Large-scale identification of phosphorylation and S-nitrosylation sites in Arabidopsis To comprehensively identify phosphorylation and S-nitrosylation sites in Arabidopsis samples, we fractionated them using high-pH reversed-phase liquid chromatography (RPLC).

[0043] The specific steps refer to Example 2, with the difference that after enzymatic digestion, there is an additional high pH reversed-phase high-performance liquid chromatography fractionation step. High pH reversed-phase high-performance liquid chromatography fractionation: Use a Waters XBridge BEH130 C18 column (3.5 μm, 4.6 × 250 mm) and an Ultimate 3000 HPLC system (Dionex, California, USA) to fractionate 3.6 mg of the mixed peptides at a flow rate of 1 mL / min. The separation uses buffer A (10 mM ammonium formate, pH 10) and buffer B (10 mM ammonium formate, containing 90% acetonitrile, pH 10), with a gradient of 60 minutes: 1% to 25% buffer B within 48 minutes, increasing to 60% within 5 minutes, increasing to 70% within 2 minutes, maintaining 70% for 5 minutes, and then returning to 1%. Monitor UV absorbance at 214 and 280 nm. Collect fractions every minute for the first 60 minutes, and then combine the 60 fractions into 12 fraction groups at equal intervals (such as fractions 1, 13, 25, 37, and 49 as a group), and then dry them under vacuum.

[0044] After high pH reversed-phase high-performance liquid chromatography fractionation, enrichment and LC-MS / MS analysis, and database search were the same as in Example 2. Our global analysis revealed a widespread phosphorylation and S-nitrosylation modification landscape in Arabidopsis seedlings, identifying 12,552 phosphorylation sites distributed in 9,230 phosphorylated peptides, of which 6,196 sites were classified as class I sites by the ptmRS algorithm. In addition, 6,108 S-nitrosylation sites were detected in 6,211 SNO peptides, of which 5,952 were classified as class I sites ( Figure 5 in A). 3,172 proteins were identified to have phosphorylation modifications, and 2,978 proteins had S-nitrosylation modifications. Among them, 968 proteins had dual modifications (simultaneous phosphorylation and S-nitrosylation modifications) ( Figure 5 in B), suggesting that the functions of these proteins may be regulated by these two post-translational modifications.

Claims

1. A method for simultaneously detecting protein phosphorylation and S-nitrosylation, characterized in that: The method comprises the following steps: 1) Labeling S-nitrosylated proteins with a phosphoaffinity tag to convert the S-nitrosylation modification into a phosphorylation mimic tag; the phosphoaffinity tag consists of three key parts: an iodoacetyl group with thiol reactivity, an alkyl spacer, and a specific phosphotag; 2) using conventional phosphoproteomics techniques to simultaneously enrich and analyze S-nitrosylated peptides and phosphorylated peptides in the enzymatic hydrolysate of the labeled protein in step 1), wherein the conventional phosphoproteomics techniques include simultaneously enriching S-nitrosylated peptides and phosphorylated peptides by metal oxide / immobilized metal ion affinity chromatography, and then performing joint detection by liquid chromatography-tandem mass spectrometry.

2. The method according to claim 1, characterized in that Step 1) The phosphate affinity tag is iodoacetamido-LC-phosphonic acid, and its structure is: .

3. The method according to claim 1, characterized in that Step 1) Before protein labeling, free thiols are blocked with iodoacetamide, and then S-nitrosylation modification is reduced with sodium ascorbate. After labeling, the protein is also reduced and alkylated before enzymatic hydrolysis. The reagents used include tris(2-carboxyethyl)phosphine / 2-chloroacetamide.

4. The method according to claim 3, characterized in that The specific steps are as follows: before protein labeling, free thiols are blocked with iodoacetamide, followed by protein precipitation to remove impurities, followed by S-nitrosylation reduction with sodium ascorbate, and labeling with 6C-CysPAT, followed by protein precipitation with acetone to remove excess 6C-CysPAT reagent; after resolubilization of the protein, disulfide bonds are reduced and blocked with tri(2-carboxyethyl)phosphine / 2-chloroacetamide, and finally the protein is enzymatically hydrolyzed into peptides by trypsin to obtain a mixture of phosphorylated peptides, PAT-labeled peptides and unmodified peptides; phosphorylated peptides and PAT-labeled peptides are simultaneously enriched by metal oxide / immobilized metal ion affinity chromatography; the enriched peptides are analyzed by liquid chromatography-tandem mass spectrometry.

5. The method according to claim 4, characterized in that Before protein labeling, free thiols were blocked with iodoacetamide, and impurities were removed by protein precipitation as follows: the protein was resuspended in HENS buffer, and iodoacetamide was added to a final concentration of 100 mM. The mixture was then incubated with shaking at 37°C for 1 hour. The mixture was vortexed and acetone was added to precipitate the protein, or methanol was added to precipitate the protein after phenol extraction. After centrifugation, the protein precipitate was washed and dried in air.

6. The method according to claim 4, characterized in that The S-nitrosylation modification was reduced with sodium ascorbate and labeled with 6C-CysPAT, and the excess 6C-CysPAT reagent was removed by acetone precipitation. The specific steps were as follows: the protein precipitate was resuspended in HENS buffer containing sodium ascorbate and 6C-CysPAT, incubated with gentle rolling at 37°C for 1 hour, and then subjected to rapid protein precipitation. After centrifugation, the protein precipitate was washed twice with 80 vol% acetone containing 10 mM NaCl and dried in air for 5 minutes to obtain the S-nitrosylation modified protein PAT-Switch.

7. The method according to claim 6, characterized in that For rapid protein precipitation, the mixture was incubated at 37°C for 1 hour with gentle rolling, mixed with 1 / 10 volume of 1 M NaCl, and incubated with 5 volumes of acetone for 30 minutes at room temperature.

8. The method according to claim 4, characterized in that The specific steps of reducing and blocking disulfide bonds with TCEP / CAA and finally digesting the protein with trypsin into peptides to obtain a mixture of phosphorylated peptides, PAT-labeled peptides and unmodified peptides are as follows: the protein precipitate after PAT-Switch labeling is resuspended in a denaturing buffer containing TCEP / CAA and incubated at 60°C for 30 minutes, and 4 volumes of methanol, 1 volume of chloroform and 3 volumes of water of the denaturing buffer are added in sequence. After centrifugation, the precipitate is washed with cold methanol containing 0.1M NH4OAc and 80vol% acetone containing 10 mM NaCl, and dried in air for 5 minutes; then resuspended in 50 mM ammonium bicarbonate buffer, 1 / 50 protein amount of trypsin is added, and incubated at 37°C for 12 hours, the digestion solution is acidified by adding formic acid to 1vol%, desalted using a peptide desalting centrifugal column, quantified using a Pierce quantitative colorimetric peptide assay kit, and vacuum dried to obtain.

9. The method according to claim 4, characterized in that The enriched peptides were analyzed by liquid chromatography-tandem mass spectrometry under the following conditions: C18 analytical column; acetonitrile gradient: 5%-25%, 0-90 min; 25%-35%, 90-105 min; 35%-85%, 105-120 min, with a flow rate of 300 nL / min; the source voltage was set to 2.0 kV, and the ion transfer tube temperature was maintained at 320 °C; the mass spectrometer was operated in data-dependent acquisition and data-independent acquisition modes; in DDA mode, MS1 scans were performed by the Orbitrap detector using 400−1300 m / z, 120 K resolution, 60% RF lens, 8E5 AGC target and 1100 ms maximum ion injection time, while MS2 scans were performed using 5.0E4 intensity threshold, 2-6 charge states, 25 s exclusion time, 54 ms maximum ion injection time, 1.6 Da isolation window, 30% HCD collision energy, 1.0E5 AGC target, and 3 s cycle time; Using the background ion (C2H6SiO)7H with m / z = 519.1388 + Perform internal calibration.

10. The method according to claim 4, characterized in that Prior to enrichment and analysis, we used high-pH reversed-phase HPLC to fractionate the peptide mixture. 3.6 mg of peptides were fractionated using a Waters XBridge BEH130 C18 column and an Ultimate3000 HPLC system at a flow rate of 1 mL / min. The separation was performed using 10 mM ammonium formate buffer A, pH 10, and 10 mM ammonium formate buffer B, pH 10, containing 90% acetonitrile. The gradient was 60 min: 1% to 25% buffer B in 48 min, increased to 60% in 5 min, increased to 70% in 2 min, maintained at 70% for 5 min, and then returned to 1%; UV absorbance was monitored at 214 and 280 nm, and fractions were collected every minute for the first 60 min. Then, 60 fractions were combined into 12 groups at equal intervals and then dried under vacuum.

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