A method for simultaneous detection of protein phosphorylation and s-nitrosylation

Abstract

This invention discloses a method for simultaneously detecting protein phosphorylation and S-nitrosylation. It utilizes phosphate affinity tags to label S-nitrosylated proteins, transforming the S-nitrosylation modification into a phosphorylation-mimicking tag. This allows for the simultaneous enrichment and analysis of both S-nitrosylation and phosphorylation using conventional phosphorylated proteomics techniques. Through testing on model proteins and complex biological samples, we have verified that this method exhibits high sensitivity, good reproducibility, and broad protein coverage, comparable to traditional single-method enrichment. It overcomes the technical challenges of analyzing modifications with different chemical properties and provides a reliable tool for comprehensive research on cross-functional PTM (phosphorylated protein-to-protein) interactions.

Description

Technical Field

[0001] This invention relates to the field of proteomics methods, specifically to a method for simultaneously detecting protein phosphorylation and S-nitrosylation. Background Technology

[0002] Post-translational modifications (PTMs) are dynamic regulators of protein function and activity. Among the more than 400 known PTMs, reversible phosphorylation factors play a central role in eukaryotic signal transduction, protein-protein interactions, enzyme activity regulation, and subcellular localization, making them the most extensively studied type of PTM. Meanwhile, protein… S S-nitrosylation (a modification that links nitric oxide (NO) molecules to cysteine ​​residues in proteins via redox reactions) has also attracted considerable attention due to its regulatory function, which is similar to phosphorylation. Recent studies have revealed the molecular mechanisms by which S-nitrosylation is widely involved in the regulation of plant growth and development and responses to abiotic stress.

[0003] Proteins typically undergo various modifications at multiple sites, and the interactions between different post-translational modifications (PTMs) add additional regulatory layers to the proteome, making it more complex. A growing body of research reveals complex crosstalk between phosphorylation and S-nitrosylation. For example, in Arabidopsis, phosphorylation of the salicylate-binding protein SABP3 requires prior S-nitrosylation, while in the BIK1 protein, phosphorylation occurs before S-nitrosylation, both jointly regulating immune signaling. This interaction is not limited to individual proteins; certain kinase PTMs can also affect the entire signaling pathway by altering the phosphorylation state of downstream substrates. Particularly noteworthy is that S-nitrosylation can modulate the activity of multiple kinases, including sucrose non-fermentation-1-related protein kinase (SnRK2), target of rapamycin (TOR), FERONIA receptor kinase (FER), mitogen-activated protein kinases (MAPKs), and thioredoxin-interacting receptor kinases (TIRKs). This indicates that the complex crosstalk between S-nitrosylation and phosphorylation plays a crucial regulatory role in plant cell signal transduction.

[0004] Although scientists are increasingly interested in the interaction between S-nitrosylation and phosphorylation, simultaneously analyzing both S-nitrosylation and phosphorylation presents technical challenges. These two modifications are present in low abundance in cells, making selective enrichment a crucial step for efficient detection using mass spectrometry (MS). However, S-nitrosylation and phosphorylation modifications have very different properties, requiring different enrichment strategies: phosphorylated proteins / peptides are usually enriched directly using metal oxide / ion affinity chromatography (MOAC / IMAC), while S-nitrosylated proteins / peptides rely on tag-switching strategies (converting S-nitrosylation modifications into other enrichable groups). For example, the traditional biotinylation technique (BST) converts S-nitrosylation into biotinylate groups. Scientists have also developed a series of new tag-switching techniques, such as isotope-encoded affinity tags (ICAT) (WuCG, 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.) and bioorthogonally cleavable Cys-BOOST (MnatsakanyanR, 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 (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 tag (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 switch tags targeting S-nitrosylation modification and phosphorylation modification have different properties, making it impossible to enrich both simultaneously. Simultaneous analysis of these two modifications requires separate or sequential enrichment and analysis, which is impractical for studies with limited sample sizes and introduces 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, simultaneous analysis of protein phosphorylation and S-nitrosylation modifications still faces technical bottlenecks. Summary of the Invention

[0005] The purpose of this invention is to provide a method for simultaneously detecting protein phosphorylation and S-nitrosylation, which solves the problem that existing technologies cannot simultaneously enrich and detect phosphorylation and S-nitrosylation.

[0006] This invention is achieved through the following technical solutions:

[0007] A method for simultaneously detecting protein phosphorylation and S-nitroso (SNO) oxidation, comprising the following steps:

[0008] 1) Label S-nitrosylated proteins with phosphorylated affinity tags (PATs) to convert S-nitrosylation modification into a phosphorylation-mimicking tag (this method is named PAT-switch); the phosphorylated affinity tag (PAT) consists of three key parts: an iodoacetyl group with thiol reactivity, an alkyl spacer, and a specific phosphate tag;

[0009] 2) Simultaneously enrich and analyze S-nitrosylated peptides (PAT-switch) and phosphorylated peptides in the enzymatic digests of the labeled protein in step 1) using conventional phosphorylated proteomics techniques. The conventional phosphorylated proteomics techniques include simultaneous enrichment of S-nitrosylated peptides (PAT-switch) and phosphorylated peptides by metal oxide / immobilized metal ion affinity chromatography (MOAC / IMAC), followed by joint detection by liquid chromatography-tandem mass spectrometry (LC-MS / MS).

[0010] Preferably, the phosphate affinity tag in step 1) is iodoacetamido-LC-phosphonic acid (6C-CysPAT), with the following structure: This is because it is not only readily available, but the thiol reactivity of its iodoacetyl group has been well-established. Furthermore, specific labeling of S-nitrosylation modifications can be achieved through pretreatment strategies such as Cys-BOOST or FAT, including blocking free thiols, reducing and labeling S-nitrosylation modifications, and reducing disulfide bonds.

[0011] Specifically, before enzymatic digestion of labeled proteins, a reduction and alkylation process for disulfide bonds is also included, using reagents including tris(2-carboxyethyl)phosphine / 2-chloroacetamide (TCEP / CAA).

[0012] S-nitrosylation modification occurs on the sulfhydryl group of cysteine ​​in proteins. Cysteine ​​exists in proteins mainly in three forms: free thiols, S-nitrosyl cysteine, and disulfide bonds. Due to their different chemical properties, they need to be treated separately. First, free thiols are blocked with iodoacetamide (IAA) before protein labeling, preserving S-nitrosylcysteine ​​and disulfide bonds. Next, impurities are removed by protein precipitation, followed by reduction of the S-nitrosylation modification with sodium ascorbate and labeling with 6C-CysPAT (converting S-nitrosylcysteine ​​to PAT-labeled cysteine). Then, the protein is precipitated with acetone to remove excess 6C-CysPAT reagent. After rehydration, the protein is reduced and disulfide bonds are blocked with TCEP / CAA. Finally, the protein is digested into peptides by trypsin, yielding a mixture of phosphorylated peptides, PAT-labeled peptides, and unmodified peptides. Phosphorylated and PAT-labeled peptides are simultaneously enriched using metal oxide / immobilized metal ion affinity chromatography, while unmodified peptides are eluted. The enriched peptides are analyzed by liquid chromatography-tandem mass spectrometry (LC-MS / MS). Because phosphorylation and PAT labeling have different mass shifts (phosphorylation is 79.97 Da, PAT labeling is 221.08 Da), mass spectrometry can clearly distinguish between these two modifications and, in conjunction with secondary mass spectrometry, can achieve peptide sequence and modification site identification, as well as quantitative analysis.

[0013] Before protein labeling, free thiols were blocked with iodoacetamide (IAA), and impurities were removed by protein precipitation. The specific steps were as follows: the protein was resuspended in HENS buffer, iodoacetamide (IAA) was added to a final concentration of 100 mM, and then incubated at 37°C with shaking (600 rpm) for 1 hour. The mixture was vortexed, and acetone was added to precipitate the protein, or the protein was extracted with phenol and then precipitated with methanol. After centrifugation, the protein precipitate was washed and dried in air.

[0014] The S-nitrosylation modification was reduced with sodium ascorbate and labeled with 6C-CysPAT. The protein was then precipitated with acetone to remove excess 6C-CysPAT reagent. The specific steps were as follows: The protein precipitate was resuspended in HENS buffer containing sodium ascorbate and 6C-CysPAT and gently incubated at 37°C for 1 hour. Then, rapid protein precipitation was performed (the mixture was mixed with 1 / 10 volume of 1M NaCl and incubated with 5 volumes of acetone for 30 minutes at room temperature). After centrifugation, the protein precipitate was washed twice with 80 vol% acetone (containing 10 mM NaCl) and air-dried for 5 minutes to obtain the S-nitrosylated protein modified by PAT-Switch.

[0015] The disulfide bonds were reduced and blocked using TCEP / CAA. Finally, the protein was digested into peptides by trypsin to obtain a mixture of phosphorylated peptides, PAT-labeled peptides, and unmodified peptides. The specific steps are as follows: The PAT-Switch labeled protein precipitate was resuspended in denaturing buffer containing TCEP / CAA and incubated at 60°C for 30 minutes. Then, 4 volumes of methanol, 1 volume of chloroform, and 3 volumes of water were added sequentially to the denaturing buffer. After centrifugation, the precipitate was washed with cold methanol (containing 0.1M NH4OAc) and 80 vol% acetone (containing 10 mM NaCl) and air-dried for 5 minutes. Then, it was resuspended in 50 mM ammonium bicarbonate buffer, and trypsin (1 / 50 of the protein weight) was added. The mixture was incubated at 37°C for 12 hours. Formic acid was added to the digestion solution to acidify it to 1 vol%. The peptide was desalted using a peptide desalting centrifuge column, quantified using a Pierce quantitative colorimetric peptide assay kit, and vacuum dried.

[0016] The enriched peptides were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS / MS) under the following conditions: separation on a C18 column using acetonitrile gradients (5%–25%, 0–90 min; 25%–35%, 90–105 min; 35%–85%, 105–120 min) at a flow rate of 300 nL / min; source voltage set to 2.0 kV; ion transfer tube temperature maintained at 320 °C; the mass spectrometer was operated in both data-dependent acquisition (DDA) and data-independent acquisition (DIA) modes. In DDA mode, MS1 scans were performed using an Orbitrap detector with a resolution of 400–1300 m / z, 120 K, 60% RF lens, 8E5 AGC target, and a maximum ion implantation time of 1100 ms, while MS2 scans were performed with an intensity threshold of 5.0E4, charge states of 2–6, an exclusion time of 25 s, a maximum ion implantation time of 54 ms, and a maximum ion implantation time of 1.6 Da. Isolation window, 30% HCD collision energy, 1.0E5 AGC target, and 3 s cycle time; using background ions (C2H6SiO)7H at m / z = 519.1388. + Perform internal calibration.

[0017] Preferably, to enhance peptide identification and data-independent acquisition (DIA) quantification, high-pH reversed-phase high-performance liquid chromatography (RP-HPLC) was used to fractionate the mixed peptides prior to enrichment and analysis. 3.6 mg of peptides were fractionated using a Waters XBridge BEH130 C18 column (3.5 μm, 4.6 × 250 mm) and an Ultimate 3000 HPLC system (Dionex, California, USA) at a flow rate of 1 mL / min. Separation was performed using buffer A (10 mM ammonium formate, pH 10) and buffer B (10 mM ammonium formate, containing 90% acetonitrile, pH 10) in a 60-minute gradient: 1% to 25% buffer B over 48 minutes, increasing to 60% over 5 minutes, increasing to 70% over 2 minutes, holding at 70% for 5 minutes, and then returning to 1%. UV absorbance was monitored at 214 and 280 nm. For the first 60 minutes, a fraction was collected every minute. The 60 fractions were then combined into 12 distillation groups at equal intervals (e.g., fractions 1, 13, 25, 37, and 49 were grouped together), and then vacuum dried.

[0018] The beneficial effects of this invention are as follows:

[0019] 1) This invention utilizes phosphorylation affinity tags (PAT) to label S-nitrosylated proteins, transforming S-nitrosylation modification into a phosphorylation-mimicking tag. This allows for the simultaneous enrichment and analysis of both S-nitrosylation and phosphorylation modifications using conventional phosphorylated proteomics techniques. Through testing on model proteins and complex biological samples, we have verified that this method is highly sensitive, reproducible, and has a broad protein coverage, comparable to traditional single enrichment methods. It overcomes the technical challenges of analyzing modifications with different chemical properties and provides a reliable tool for comprehensive research on cross-linking of PTM.

[0020] 2) This invention significantly improves the reproducibility and cost-effectiveness of experiments by acquiring dual-modified omics datasets within the same workflow. Compared to parallel or sequential single-omics analyses, this method reduces manpower, reagent consumption, and instrument usage time, while the consistency between the two omics datasets provides a more reliable basis for biological research.

[0021] 3) More importantly, this method provides a powerful tool for studying plant signal transduction, stress response, and metabolic regulation. We have used it to discover many new S-nitrosylation sites and revealed potential PTM interactions in key regulatory proteins. Attached Figure Description

[0022] Figure 1 This is a schematic diagram of the process flow of the present invention;

[0023] Figure 2 This is a graph showing the enrichment capacity of the phosphate column for S-nitrosolated BSA-digested peptides in Example 1. A is the mass spectrum of the untreated group 3 sample; B is the mass spectrum of the unenriched group 2 sample; C is the mass spectrum of the group 1 sample; and D is the sensitivity evaluation graph for trace S-nitrosolated BSA (100 fmol).

[0024] Figure 3 This is a feasibility verification diagram for the analysis of protein phosphorylation and S-nitrosylation in biological samples in Example 2; where A is a comparison of peptide coverage with single-omics technology; and B is the number of phosphorylated peptides identified before and after PAT treatment.

[0025] Figure 4 This is a reproducibility evaluation of the analysis of protein phosphorylation and S-nitrosylation in biological samples in Example 2; where A is a comparison of total ion chromatograms (TIC) of three parallel experiments; B and C are scatter plots of peptide abundance among replicates of data-dependent acquisition mode (DDA) and data-independent acquisition mode (DIA), respectively; and D is the correlation between biological replicates.

[0026] Figure 5This is a statistical analysis of the large-scale identification of phosphorylation and S-nitrosylation sites in Arabidopsis thaliana in Example 3, where A is the total number of modified polypeptides and sites identified; and B is the number of phosphorylated, S-nitrosylated modified proteins and dual-modified proteins identified. Detailed Implementation

[0027] The following is a further description of the invention, but not a limitation thereof.

[0028] Reagents and materials:

[0029] Bovine serum albumin (BSA), iodoacetamide (IAA), 2-chloroacetamide (CAA), dithiothreitol (DTT), tris(2-carboxyethyl)phosphine (TCEP), acetone, and chloroform used in the experiments were purchased from Sigma-Aldrich (Missouri, USA). LC-MS grade acetonitrile (ACN), water, methanol, formic acid (FA), iodoacetamide-LC-phosphonic acid (6C-CysPAT), protease and phosphatase inhibitor mini-tablets, dicaprylic acid (BCA) protein assay kit, Zeba protein desalting centrifuge column, and Pierce™ peptide desalting centrifuge column were purchased from Thermo Fisher Scientific (Massachusetts, USA). HEPES, EDTA, copper hydroxide reagent, sucrose, sodium dodecyl sulfate (SDS), ammonium acetate (NH4OAc), sodium chloride (NaCl), sodium nitrite (NaNO2), hydrochloric acid (HCl), sodium ascorbate, urea, and ammonium bicarbonate (NH4HCO3) were purchased from Aladdin Scientific (Shanghai, China). Sequencing-grade trypsin was supplied by Promega Corporation (Wisconsin, USA).

[0030] The mixing mill (MM400) and ultrasonic homogenizer (Bioruptor UCD-300) were purchased from RETSCH (Germany) and Diagenode (Belgium), respectively. The centrifuge (Multifuge 1S-R), nano-level liquid chromatograph (Ultimate™ 3000RSLCnano), and mass spectrometer (Orbitrap Fusion) were from Thermo Fisher Scientific (San Jose, California, USA).

[0031] Example 1: Validating the enrichment effect using model proteins

[0032] To evaluate its ability to enrich PAT-labeled peptides, we used artificially S-nitrosolated bovine serum albumin (S-nitrosolated BSA) as a model protein.

[0033] The preparation of artificial S-nitrosolated bovine serum albumin (S-nitrosolated BSA) was as follows: Following 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-nitrosolated with sodium nitrite to prepare artificial S-nitrosolated BSA. In simple terms, 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 Then, the solution was reduced with 20 mM TCEP at 37 °C for 30 minutes. After two rounds of desalting using a Zeba centrifuge column, the pH was adjusted to 1.0 with HCl, and the solution was mixed with an equal volume of NaNO2 (approximately 20 molar excess relative to the BSA thiol). The mixture was incubated in the dark at 25 °C for 1 hour. Finally, precipitation was carried out with 4 volumes of cold acetone at -20 °C for 2 hours, followed by washing twice with cold acetone and methanol.

[0034] The prepared S-nitrosylated BSA samples were divided into three groups, designated as Group 1 to Group 3. Group 1 samples underwent the following treatment: free thiols were blocked with iodoacetamide (IAA), labeled with PAT-Switch, followed by protein reduction, alkylation, and enzymatic digestion for enrichment. Group 2 samples were treated similarly to Group 1 samples, except that no enrichment was performed. Group 3 samples were treated similarly to Group 1 samples, except that no PAT-Switch labeling or enrichment was performed; these served as a blank control. After treatment, the proteins were enzymatically digested into peptides and then analyzed by liquid chromatography-tandem mass spectrometry.

[0035] The specific process for processing Group 1 samples is as follows:

[0036] 1) Blocking free thiols in S-nitrosolated bovine serum albumin (S-nitrosolated BSA) with iodoacetamide (IAA): 1 mg of artificial S-nitrosolated bovine serum albumin (S-nitrosolated BSA) was resuspended in 500 μL HENS buffer (100 mM HEPES, pH 7.7, 1 mM EDTA, 0.1 mM copper reagent, 1 wt% SDS). Iodoacetamide (IAA) was added to a final concentration of 100 mM, followed by incubation at 37°C with shaking (600 rpm) for 1 hour. The mixture was then mixed with 50 μL of 1 M NaCl and incubated with 2.5 mL of acetone at room temperature for 30 minutes. After centrifugation (8,000 × g, 10 min, 4°C), the protein precipitate was washed twice with 80 vol% acetone (containing 10 mM NaCl) and air-dried for 5 minutes.

[0037] 2) Reduction of S-nitrosylation modification and PAT-Switch labeling: All operations were performed under low light conditions. The protein precipitate obtained in step 1) was resuspended in 500 μL HENS buffer (containing 20 mM sodium ascorbate and 5 mM 6C-CysPAT). The mixture was shaken at 37°C for 1 hour, followed by rapid protein precipitation (the mixture was mixed with 50 μL 1 M NaCl and incubated with 2.5 mL acetone for 30 minutes at room temperature). After centrifugation (8,000 × g, 10 min, 4°C), the protein precipitate was washed twice with 80 vol% acetone (containing 10 mM NaCl) and air-dried for 5 minutes to obtain PAT-Switch labeled S-nitrosylated protein. (This step was skipped for the untreated control group 3 samples).

[0038] 3) Reduction, alkylation, and enzymatic digestion of PAT-Switch labeled S-nitrosylated protein: The protein precipitate (PAT-Switch labeled S-nitrosylated protein) obtained in step 2) was resuspended in 500 μL denaturing buffer (8 M urea, 50 mM ammonium bicarbonate, 10 mM TCEP, and 40 mM CAA) and incubated at 60°C for 30 min. Precipitation was performed using the chloroform-methanol method, with 2 mL of methanol, 0.5 mL of chloroform, and 1.5 mL of water added sequentially. After centrifugation (8,000 × g, 10 min, 4°C), the precipitate was washed with cold methanol (containing 0.1 M NH4OAc) and 80 vol% acetone (containing 10 mM NaCl) and air-dried for 5 min. The precipitate was resuspended in 500 μL of 50 mM ammonium bicarbonate buffer, and trypsin (1 / 50 of the protein weight) was added. The mixture was incubated at 37°C for 12 hours. Formic acid was added to the digest to a final concentration of 1 vol% for acidification. The peptide was desalted using a peptide desalting centrifuge column, quantified using a Pierce Quantitative Peptide Assay Kit, and then vacuum dried.

[0039] 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 phosphate capture column for online enrichment. After washing away non-specifically bound peptides with loading buffer, the target peptides were eluted with 1 M NH4H2PO4 and subsequently captured on a downstream C18 capture column (samples in groups 2-3 do not require enrichment with a phosphate 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 min) 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 both data-dependent acquisition (DDA) and data-independent acquisition (DIA) modes. In DDA mode, the MS1 scan was performed using an Orbitrap detector with 400–1300 m / z, 120 K resolution, 60% RF lens, 8E5 AGC target, and a 1100 ms maximum ion implantation time, while the MS2 scan used a 5.0E4 intensity threshold, 2–6 charge states, 25 s exclusion time, 54 ms maximum ion implantation time, 1.6 Da isolation window, 30% HCD collision energy, 1.0E5 AGC target, and a 3 s cycle time. Background ions (C2H6SiO)7H were used at m / z = 519.1388. +Perform internal calibration (to lock in quality).

[0040] 5) Database Search: Peptide identification and label-free quantification (LFQ) were performed using Proteome Discoverer™ 3.1 software and the SEQUEST search engine. The original file was searched against the BSA FASTA file (P02769 in Swiss-Prot). Trypsin was selected as the enzyme, allowing a maximum of two missed cleavage sites. The precursor quality tolerance was set to 10 ppm, and the fragment quality tolerance was set to 0.02 Da. Variable modifications included 6C-CyaPAT (C), oxidation (M), and carbamoyl methylation (C), with a maximum of five modifications allowed per peptide. The false discovery rate (FDR) of the peptides was calculated using the Percolator node algorithm, with high-confidence and medium-confidence thresholds of 0.01 and 0.05, respectively.

[0041] Mass spectrometry analysis showed that in the unenriched S-nitrosylated BSA hydrolysis products, the unmodified peptides had a strong signal, while the S-nitrosylated peptides had a weak signal. Figure 2 In section A), PAT labeling alone did not significantly improve the detection of S-nitrosyl peptides without enrichment. Figure 2 The B in the figure indicates limited modification efficiency. However, the PAT labeling and enrichment analysis of group 1 samples significantly enhanced the signal of the PAT-labeled peptides (B). Figure 2 (C in BSA). We identified 30 S-nitrosylation 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 process using trace amounts of S-nitrosylated BSA (100 fmol) detected 7 PAT-labeled peptides (C in BSA). Figure 2 The high specificity and sensitivity of the method are demonstrated by the D in the figure.

[0042] Example 2: Testing method performance in complex biological samples

[0043] After validating 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 techniques—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. Plant2024, 17(1): 199-213.], the former for S-nitrosoproteomics and the latter for phosphorylated proteomics. We used Arabidopsis thaliana seedling samples, processed them using the PAT-switch method, and performed three single injections for online enrichment and LC-MS / MS analysis.

[0044] The specific steps are as follows:

[0045] 1) Arabidopsis thaliana whole protein extraction and free thiol blocking treatment: All operations were performed under low temperature and low light conditions using metal-free reagents (unless otherwise specified). First, Arabidopsis thaliana seedlings were ground into a fine powder in liquid nitrogen using a mixing grinder. 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 copper reagent, 1 wt% SDS), which also contained 0.9 M sucrose and was supplemented with protease / phosphatase inhibitors. Protein was extracted by sonication for 10 cycles (10 seconds on / 10 seconds off) in an ice bath. The homogenate was centrifuged at 16,000 × g, 4°C for 15 minutes, and the supernatant was collected. Iodoacetamide (IAA) was added to a final concentration of 100 mM, followed by incubation at 37°C with shaking (600 rpm) for 1 hour. Mix the solution with an equal volume of Tris-saturated phenol (pH 8.0), vortex for 10 min, and centrifuge at 8,000 × g, 4 °C for 20 min. Collect the upper phenol phase, mix with 5 volumes of pre-cooled methanol (containing 0.1 M NH4OAc), and incubate at -20 °C for 3 h. Precipitate the protein by centrifugation (8,000 × g, 20 min, 4 °C), wash with cold methanol (containing 0.1 M NH4OAc) and 80 vol% acetone (containing 10 mM NaCl), and air dry for 5 min.

[0046] 2) Reduction of S-nitrosylation modification and PAT labeling: The protein precipitate obtained in step 1) was resuspended in 500 μL HENS buffer containing 20 mM sodium ascorbate and 5 mM 6C-CysPAT. The mixture was incubated with shaking at 37°C for 1 hour. The mixture was vortexed with 50 μL 1 M NaCl and incubated with 2.5 mL acetone at room temperature for 30 minutes. After centrifugation (8,000 × g, 10 min, 4°C), the protein precipitate was washed twice with 80 vol% acetone (containing 10 mM NaCl) and air-dried for 5 minutes.

[0047] 3) Protein reduction, alkylation, and enzymatic digestion after PAT-Switch labeling: The protein precipitate obtained in step 2) was resuspended in denaturing buffer (8 M urea, 50 mM ammonium bicarbonate, 10 mM TCEP, and 40 mM CAA) and incubated at 60°C for 30 min. Four volumes of methanol, one volume of chloroform, and three volumes of water were added sequentially to the denaturing buffer. After centrifugation (8,000 × g, 10 min, 4°C), the precipitate was washed with cold methanol (0.1 M NH4OAc) and 80 vol% acetone (containing 10 mM NaCl) and air-dried for 5 min. The precipitate was resuspended in 500 μL of 50 mM ammonium bicarbonate buffer, trypsin (1 / 50 of the protein volume) was added, and the mixture was incubated at 37°C for 12 h. The digestion solution was acidified with formic acid to 1 vol%, desalted using a peptide desalting centrifuge column, quantified using a Pierce quantitative colorimetric peptide assay kit, and vacuum dried.

[0048] 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 phosphate capture column for online enrichment. After washing away non-specifically bound peptides with loading buffer, the target peptides were eluted with 1 M NH4H2PO4 and subsequently captured on a downstream 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%–25%, 0–90 min; 25%–35%, 90–105 min; 35%–85%, 105–120 min) 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 both data-dependent acquisition (DDA) and data-independent acquisition (DIA) modes. In DDA mode, the MS1 scan was performed using an Orbitrap detector with a resolution of 400–1300 m / z, 120 K, 60% RF lens, 8E5 AGC target, and a maximum ion implantation time of 1100 ms, while the MS2 scan was performed with an intensity threshold of 5.0E4, 2–6 charge states, a 25 s exclusion time, a maximum ion implantation time of 54 ms, a 1.6 Da isolation window, 30% HCD collision energy, 1.0E5 AGC target, and a 3 s cycle time. Background ions (C2H6SiO)7H were used at m / z = 519.1388. + Perform internal calibration (to lock in quality).

[0049] 5) Database Search: Peptide identification and label-free quantification (LFQ) were performed using Proteome Discoverer™ 3.1 software and the SEQUEST search engine. The original files were searched against FASTA files of the Arabidopsis proteome (TAIR10, EnsemblPlants). Trypsin was selected as the enzyme, allowing a maximum of two missed cleavage sites. The precursor quality tolerance was set to 10 ppm, and the fragment quality tolerance was set to 0.02 Da. Variable modifications included 6C-CyaPAT (C), phosphorylation (S, T, Y), oxidation (M), and carbamoyl methylation (C), with a maximum of five modifications allowed per peptide. The false discovery rate (FDR) of peptides was calculated using the Percolator node algorithm, with high-confidence and medium-confidence thresholds of 0.01 and 0.05, respectively. The phosphorylation and S-nitrosylation probabilities at each site were assessed using ptmRS, with a p-value cutoff of 0.75; sites above this were classified as Class I confidence sites.

[0050] This method identified 2,442 S-nitrosylated peptides and 4,959 phosphorylated peptides from Arabidopsis seedlings (Figure 3, A). When using 300 μg of protein, the coverage of phosphorylated peptides was slightly lower than GreenPhos (5,703), while the S-nitrosylation performance was comparable to FAT (2,559). 4,201 phosphorylated peptides were identified in untreated samples (i.e., conventional phosphorylated proteomics analysis), while 4,465 phosphorylated peptides were identified in PAT-treated samples. Figure 3 The B in the figure indicates that PAT labeling does not interfere with phosphorylation analysis. Furthermore, increasing the LC-MS / MS gradient time from 120 minutes to 240 minutes only resulted in a slight increase in the number of phosphorylated peptides identified. Figure 3 (B in the text) indicates that a gradient of 120 minutes is a suitable analytical condition.

[0051] Quantitative reproducibility was assessed by repeat analysis; the base peak chromatograms from three identical runs were similar. Figure 4 (A in Figure 4). For parallel enrichment and LC-MS / MS analysis, the Pearson correlation coefficients for the intensity of modified peptides between replicates were high, reaching 0.982 and 0.973 in DDA and DIA modes, respectively (B in Figure 4). Figure 4 C in the text). Biological replicates across the entire workflow showed excellent reproducibility, with a Pearson correlation coefficient of 0.967 (C). Figure 4 (D in the text). In contrast, GreenPhos reported correlation coefficients of 0.97 and 0.95 (data from the literature), indicating a slight improvement in the reproducibility of the new method.

[0052] Example 3: Large-scale identification of phosphorylation and S-nitrosylation sites in Arabidopsis thaliana

[0053] To comprehensively identify phosphorylation and S-nitrosylation sites in Arabidopsis thaliana samples, we used high-pH reversed-phase liquid chromatography (RPLC) for fractionation.

[0054] The specific steps are the same as in Example 2, except that a high-pH reversed-phase high-performance liquid chromatography (RP-HPLC) fractionation step is added after enzymatic hydrolysis. High-pH RPI fractionation: 3.6 mg of the mixed peptides were fractionated using a Waters XBridge BEH130 C18 column (3.5 μm, 4.6 × 250 mm) and an Ultimate 3000 HPLC system (Dionex, California, USA) at a flow rate of 1 mL / min. Separation was performed using buffer A (10 mM ammonium formate, pH 10) and buffer B (10 mM ammonium formate, containing 90% acetonitrile, pH 10) with a gradient over 60 minutes: 1% to 25% buffer B over 48 minutes, increasing to 60% over 5 minutes, increasing to 70% over 2 minutes, maintaining 70% for 5 minutes, and then returning to 1%. UV absorbance was monitored at 214 and 280 nm. For the first 60 minutes, a fraction was collected every minute. Then, the 60 fractions were combined into 12 distillation groups at equal intervals (e.g., fractions 1, 13, 25, 37, and 49 were grouped together) and then vacuum dried.

[0055] Enrichment and LC-MS / MS analysis were performed after high-pH reversed-phase high-performance liquid chromatography fractionation, and database searching was conducted 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 across 9,230 phosphorylated peptides, of which 6,196 sites were classified as Class I sites using the ptmRS algorithm. Furthermore, 6,108 S-nitrosylation sites were detected in 6,211 SNO peptides, of which 5,952 were classified as Class I sites. Figure 5 (A) 3172 proteins were identified as undergoing phosphorylation modification, and 2978 proteins as undergoing S-nitrosylation modification. Among these, 968 proteins underwent dual modification (simultaneous phosphorylation and S-nitrosylation modification). Figure 5 The B in the text suggests that the function of these proteins may be regulated by two post-translational modifications.

Claims

1. A method for simultaneous detection of protein phosphorylation and S-nitrosylation, characterized by, The method comprises the following steps: 1) protein labeling before free thiols are blocked with iodoacetamide, followed by removing impurities by precipitating protein, the specific steps are as follows: the protein is resuspended in HENS buffer, iodoacetamide is added to a final concentration of 100 mM, followed by incubation at 37°C for 1 hour with shaking, the mixture is vortexed, and the protein is precipitated by adding acetone or using phenol to extract the protein, followed by adding methanol to precipitate the protein, after centrifugation, the protein precipitate is washed and dried in air; S-nitrosylation modification is reduced with sodium ascorbate, and S-nitrosylated protein is labeled with a phospho affinity tag, and S-nitrosylation modification is converted into a phospho mimic tag; the phospho affinity tag consists of three key parts: an iodoacetyl group with thiol reactivity, an alkyl spacer, and a specific phospho tag; the phospho affinity tag is iodoacetamido-LC-phosphonic acid, and its structure is: ; then the protein is precipitated with acetone to remove excess 6C-CysPAT reagent; after resuspension of the protein, tris(2-carboxyethyl)phosphine / 2-chloroacetamide is used to reduce and block disulfide bonds, and finally the protein is digested into peptide segments by trypsin to obtain a mixture of phosphorylated peptides, PAT-labeled peptides, and unmodified peptides; 2) Simultaneously enrich and analyze the S-nitrosylated peptides and phosphorylated peptides in the proteolytic products of the labeled proteins in step 1) after trypsin digestion using conventional phosphorylation proteomics technology, which comprises simultaneous enrichment of S-nitrosylated peptides and phosphorylated peptides by immobilized metal ion affinity chromatography, and then combined detection by liquid chromatography-tandem mass spectrometry; the immobilized metal ion affinity chromatography uses a phosphoric acid capture column; Before enrichment and analysis, the mixed peptides are fractionated by high-pH reverse-phase high-performance liquid chromatography, 3.6 mg of peptides are fractionated using a Waters XBridge BEH130 C18 column and an Ultimate 3000 HPLC system at a flow rate of 1 mL / min, the separation is performed using 10 mM ammonium formate buffer A at pH 10 and 10 mM ammonium formate buffer B containing 90% acetonitrile at pH 10, the gradient is 60 minutes: 1% to 25% buffer B in 48 minutes, increased to 60% in 5 minutes, increased to 70% in 2 minutes, maintained at 70% for 5 minutes, and then returned to 1%; the UV absorbance is monitored at 214 and 280 nm, and the fractions are collected once every minute for the first 60 minutes, and then 60 fractions are equally combined into 12 fraction groups, and then vacuum dried.

2. The method of claim 1, wherein, The S-nitrosylated modification is reduced by sodium ascorbate and labeled with 6C-CysPAT, and the excess 6C-CysPAT reagent is removed by acetone precipitation, and the specific steps are as follows: the protein precipitate is resuspended in HENS buffer containing sodium ascorbate and 6C-CysPAT, incubated at 37°C with gentle rolling for 1 hour, then rapid protein precipitation is performed, after centrifugation, the protein precipitate is washed twice with 80vol% acetone containing 10 mM NaCl, and dried in air for 5 minutes to obtain the protein with S-nitrosylated modification being PAT-switched.

3. The method of claim 2, wherein, The method for rapid protein precipitation is as follows: mix the mixed solution incubated at 37°C with gentle rolling for 1 hour with 1 / 10 volume of 1M NaCl, and incubate with 5 volumes of acetone at room temperature for 30 minutes.

4. The method of claim 1, wherein, The specific steps of the mixture of phosphorylated peptides, PAT labeled peptides and unmodified peptides obtained by reducing and blocking disulfide bonds with TCEP / CAA and finally enzymatic digestion of the protein into peptides are as follows: resuspend the PAT-Switch labeled protein precipitate in denaturing buffer containing TCEP / CAA and incubate at 60°C for 30 minutes, sequentially add 4-fold volume of methanol, 1-fold volume of chloroform and 3-fold volume of water to the denaturing buffer, after centrifugation, the precipitate is washed with 0.1M NH4OAc cold methanol and 80vol% acetone containing 10mM NaCl, and dried in air for 5 minutes; then resuspend in 50mM ammonium bicarbonate buffer, add 1 / 50 protein amount of trypsin, and incubate at 37°C for 12 hours, add formic acid to 1vol% acidification to the digestion solution, desalt with a peptide desalting column, quantify with a Pierce quantitative colorimetric peptide assay kit, and vacuum dry to obtain.

5. The method of claim 1, wherein, The enriched peptides are analyzed by liquid chromatography-tandem mass spectrometry under the following conditions: C18 analysis column; acetonitrile gradient: 5%-25%, 0-90 minutes; 25%-35%, 90-105 minutes; 35%-85%, 105-120 minutes, flow rate 300 nL / min; source voltage set to 2.0 kV, ion transfer tube temperature maintained at 320°C; mass spectrometer operates in data-dependent acquisition and data-independent acquisition modes; in DDA mode, MS1 scanning is detected by Orbitrap detector using 400-1300 m / z, 120K resolution, 60% RF lens, 8E5 AGC target and 1 100 ms maximum ion injection time, while MS2 scanning uses 5.0E4 intensity threshold, 2-6 charge state, 25s exclusion time, 54 ms maximum ion injection time, 1.6 Da isolation window, 30% HCD collision energy, 1.0E5 AGC target and 3s cycle time; Background ion (C2H6SiO)7H at m / z = 519.1388 was used + Internal calibration was performed.

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