Isothiocyanate target identification method based on proteolysis-assisted cyclization reaction
By employing protein hydrolysis-assisted cyclization reaction and click chemistry-based mass spectrometry, the false positive problem in target identification during isothiocyanate (ITC) proteomics analysis was solved, achieving highly sensitive site-level target identification, especially for the specific capture and quantitative verification of low-abundance targets.
Patent Information
- Application Number
- CN202511292451.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-11
- Publication Date
- 2025-11-14
AI Technical Summary
In existing technologies, the poor probe specificity or unstable adducts in isothiocyanate (ITC) proteomics analysis result in a high false positive rate for target identification and the inability to achieve site-level identification.
A protein hydrolysis-assisted cyclization reaction was employed, in which an isothiocyanate chemical probe reacted with protein cysteine residues to form a stable N-terminal dihydrothiazolide adduct. Target identification was then performed using click chemistry and mass spectrometry techniques.
It improves target coverage, reduces the risk of false positives, and achieves highly sensitive site-level target identification, especially for the specific capture and quantitative verification of low-abundance targets.
Smart Images
Figure CN120948672A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of chemical proteomics technology, specifically to a method for identifying isothiocyanate targets based on protein hydrolysis-assisted cyclization reactions. Background Technology
[0002] Isothiocyanates (ITCs, -N=C=S) are a unique class of electrophilic agents, abundant in cruciferous vegetables such as broccoli, kale, cabbage, and mustard. Studies have shown that these natural products contribute to the chemopreventive activity of these vegetables. Furthermore, both natural and synthetic ITCs exhibit a variety of biological effects, including anti-carcinogenic responses, redox regulation, bacterial defense, and apoptosis induction. The mechanism of action (MoA) of ITCs is largely attributed to their unique chemical reactivity, which allows them to form thioacryl adducts with protein cysteine thiols. To date, most ITC target identification methods are target-specific, using recombinant or in vitro isolated proteins for target identification. Therefore, developing ITC target identification methods at the proteomics level would be highly significant.
[0003] Most mass spectrometry (MS)-based chemical proteomics methods can be used to analyze the interactions between small molecules and the proteome. Among these, affinity-based (or compound-centric) methods typically rely on chemical probes of the bioactive compound under investigation to enrich small molecule targets from the biological system. For example, Chung and colleagues used radiolabeled or biotinylated probes to identify dozens of potential protein targets for ITC compounds, but did not report specific modification sites. A major limitation of this protein-level identification is the false positives caused by nonspecific affinity trapping.
[0004] Activity-based protein analysis (ABPP) has become the preferred method for site-specific target analysis of electrophilic small molecules (such as covalent ligands, lipid peroxidation products, tumor metabolites, and natural products). In a typical ABPP procedure, a thiol-reactive probe of iodoacetamide-alkyne (IA) "competes" with the electrophilic reagent under study; a decrease in probe reactivity indicates the presence of the target. A similar ABPP method was used to perform a global analysis of cysteine targets of Arabidopsis thaliana ITC compounds in plant pathogens, revealing that HrpSC209 is a functional target regulating plant defense.
[0005] While existing iodoacetamide probes can react with cysteine residues, their reaction kinetics and target preferences may differ from other types of electrophilic reagents, leading to errors in quantitative results and poor specificity. Furthermore, ITC-derived thioacyl adducts are susceptible to hydrolysis and transacylation reactions, posing a significant challenge to the direct characterization of ITC-cysteine interactions. In addition, the instability of ITC-cysteine adducts makes it difficult to directly identify ITC-derived modifications at the site level using proteomics. Therefore, developing ITC-based analytical probes is essential for target identification of natural products. In summary, there is an urgent need to propose an isothiocyanate target identification method based on proteolysis-assisted cyclization reactions that can stabilize ITC-cysteine adducts and achieve global target analysis and identification. Summary of the Invention
[0006] The purpose of this invention is to provide a method for isothiocyanate target identification based on protein hydrolysis-assisted cyclization reaction, in order to overcome the shortcomings of existing technologies in isothiocyanate (ITC) proteomics analysis, which suffer from high false positive rates in target identification due to poor probe specificity or unstable adducts, and cannot achieve site-level identification.
[0007] To achieve the above objectives, the present invention provides the following technical solution: a method for identifying isothiocyanate targets based on protein hydrolysis-assisted cyclization reaction, specifically including the following steps:
[0008] S1. Incubate the isothiocyanate-containing chemical probe with the biological sample, and then react the isothiocyanate-containing chemical probe with the protein cysteine residues in the biological sample to obtain isothiocyanate-labeled protein.
[0009] S2. The isothiocyanate-labeled protein is digested by protease to obtain a short peptide containing an N-terminus. Then, a proteolytic cyclization reaction of the short peptide containing an N-terminus is triggered to obtain an isothiocyanate-modified peptide.
[0010] S3. The isothiocyanate-modified peptide is coupled with a biotinylated reagent through a click chemical reaction, and then the coupling product is captured by streptavidin and photodegraded by ultraviolet light to finally obtain a cysteine adduct.
[0011] S4. The cysteine adduct was analyzed by liquid chromatography-tandem mass spectrometry, and the analysis results were then analyzed using computational tools to identify the cysteine adduct sites and their corresponding protein targets.
[0012] Furthermore, the isothiocyanate-containing chemical probe in S1 is an isothiocyanate analog containing an azide group or an alkyne group.
[0013] Furthermore, the isothiocyanate analogue is propargyl isothiocyanate or an alkyne derivative of sulforaphane.
[0014] Furthermore, the biological sample in S1 is at least one of cell lysate, tissue extract, or in vitro cultured intact cells.
[0015] Furthermore, the protease in S2 is at least one of trypsin, LysC, and proteinase K.
[0016] Furthermore, the protease hydrolysis is a tandem digestion, comprising the following steps:
[0017] A1. First, the isothiocyanate-labeled protein was alkylated with iodoacetamide, and then restricted hydrolysis was performed using proteinase K to obtain a long peptide containing isothiocyanate adduct.
[0018] A2. Subsequently, a second digestion was performed using trypsin or LysC to cleave the long peptide containing the isothiocyanate adduct, yielding a short peptide with an N-terminus.
[0019] Furthermore, the biotinylation reagent in S3 is either light azide-UV cleavable biotin or diazo-UV cleavable biotin.
[0020] Furthermore, the calculation tool in S4 is a modification site analysis software based on mass spectrometry data, used for the precise mass shift of cysteine adducts and their corresponding peptide sequences.
[0021] Compared with existing technologies, the isothiocyanate target identification method based on protein hydrolysis-assisted cyclization reaction provided by this invention has the following advantages:
[0022] 1. This invention develops a chemical proteomics method based on activity and affinity through protein hydrolysis-assisted cyclization (PAC), which enables chemical proteomics identification of ITC targets at the site level. It transforms the traditional unstable thioyl adduct formed by isothiocyanate (ITC) and cysteine into a stable N-terminal dihydrothiazolide peptide adduct, solving the problem of mass spectrometry signal loss caused by adduct hydrolysis / transamination in ITC target identification, and providing a chemically stable basis for subsequent target analysis.
[0023] 2. The target coverage of the method of this invention is improved by 138%, and 3056 cysteine sites are identified by tandem digestion, which is far higher than the 1282 sites identified by trypsin digestion alone. Among them, the proportion of non-K / R adjacent sites reaches 89%, while the traditional method is ≤32%. Modification sites are directly analyzed by LC-MS / MS and the computational tool pChem, avoiding the false positive risk of traditional methods that can only identify protein levels.
[0024] 3. The method of the present invention has high sensitivity, based on click chemistry enrichment and light / heavy isotope labeling, to achieve specific capture and quantitative verification of low abundance targets.
[0025] 4. The method of this invention combines ITC target identification with functional verification. Taking sulforaphane as an example, benchmark tests were conducted to identify its target, revealing that C67 of PLK1 is one of the functional targets of SFN. This method can be applied to the target identification of such unique electrophilic reagents, providing precise site information for drug design targeting cysteine. Attached Figure Description
[0026] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in this invention. For those skilled in the art, other drawings can be obtained based on these drawings.
[0027] Figure 1 A diagram illustrating the ITCyne probe-driven adduct mechanism provided in this embodiment of the invention;
[0028] Figure 2 The protein hydrolysis-assisted cyclization technology provided in this embodiment of the invention facilitates the chemical proteomics identification of ITC targets.
[0029] Figure 3 A chemical proteomics diagram illustrating the mechanism of action of sulforaphane provided in this embodiment of the invention. Detailed Implementation
[0030] To enable those skilled in the art to better understand the technical solution of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings.
[0031] Example 1:
[0032] The method for isothiocyanate target identification based on protein hydrolysis-assisted cyclization reaction includes the following steps:
[0033] S1. Incubate a biological sample with a chemical probe containing isothiocyanate, and then react the chemical probe containing isothiocyanate with cysteine residues of proteins in the biological sample to obtain isothiocyanate-labeled proteins; the chemical probe containing isothiocyanate is an isothiocyanate analog containing an azide group or an alkyne group; the isothiocyanate analog is propargyl isothiocyanate or an alkyne derivative of sulforaphane; the biological sample is at least one of cell lysate, tissue extract, or in vitro cultured intact cells.
[0034] The specific implementation method involves incubating a chemical probe containing isothiocyanate with cell lysate, and then reacting the chemical probe containing isothiocyanate with protein cysteine residues in the cell lysate to obtain an isothiocyanate-labeled protein sample; wherein the chemical probe containing isothiocyanate is propargyl isothiocyanate or an alkyne derivative of sulforaphane.
[0035] S2. The isothiocyanate-labeled protein is digested by protease to obtain a short peptide containing an N-terminus, and then a proteolytic cyclization reaction of the short peptide containing an N-terminus is triggered to obtain an isothiocyanate-modified peptide; the protease is at least one of trypsin, LysC, and proteinase K.
[0036] Proteolytic digestion is a tandem digestion process, including the following steps:
[0037] A1. First, the isothiocyanate-labeled protein was alkylated with iodoacetamide to protect non-target cysteine residues and reduce non-specific binding. Then, it was subjected to restrictive hydrolysis using proteinase K to obtain a long peptide containing isothiocyanate adduct.
[0038] A2. Subsequently, a second digestion was performed using trypsin or LysC to cleave the long peptide containing the isothiocyanate adduct, yielding a short peptide with an N-terminus.
[0039] S3. The isothiocyanate-modified peptide is coupled with a biotinylation reagent via a click chemical reaction. The coupling product is then captured using streptavidin and photolyzed under ultraviolet light to obtain a cysteine adduct. The biotinylation reagent is azide-UV cleavable-biotin and azido-UV cleavable-biotin.
[0040] The specific implementation method involves coupling the isothiocyanate-modified peptide with commercially available probes light (L) azido-UV cleavable-biotin and heavy (H) azido-UV cleavable-biotin (Az-UV-Biotin) reagents via click chemistry. The two are then mixed in a 1:1 ratio, and the biotinylated peptide is captured using streptavidin and photodegraded under 365 nm UV light irradiation to finally obtain a cysteine adduct.
[0041] S4. Cysteine adducts were analyzed by liquid chromatography-tandem mass spectrometry, and the analysis results were then analyzed using a computational tool to identify the cysteine adduct sites and their corresponding protein targets. The computational tool was a modification site analysis software based on mass spectrometry data, used for the precise mass shift of cysteine adducts and their corresponding peptide sequences.
[0042] The specific implementation method involves analyzing cysteine adducts using liquid chromatography-tandem mass spectrometry (LC-MS / MS), then interpreting the analysis results using the MS database and pChem calculation tool to identify the precise mass shift of the cysteine adduct and its corresponding peptide sequence, identifying isotope-labeled peptides, thereby obtaining the cysteine adduct site. Based on the modification site analysis results, peptides containing cysteine adduct sites are matched to the corresponding proteins to determine the protein targets corresponding to the cysteine adduct sites.
[0043] Example 2:
[0044] Using propargyl isothiocyanate (ITCyne) to investigate the reactivity of ITC-based warheads in the natural proteome;
[0045] Figure 1 A diagram illustrates the workflow for identifying ITCyne adduction sites in the cysteine genome. The specific steps are as follows:
[0046] S1. First, 100 μM MITCyne was incubated with cell lysate for 4 h, followed by alkylation with iodoacetamide (IA) and digestion with trypsin.
[0047] S2, then through Cu I Catalytic cycloaddition reaction (CuAAC) was used to react the peptide labeled with propargyl isothiocyanate probe with commercial probes light (L) and heavy (H) azide-UV cleavable-biotin (Az-UV-Biotin) reagents, and the peptides and biotin reagents were mixed in equal volumes at a 1:1 ratio.
[0048] S3. Then, streptavidin is used to capture and enrich the biotinylated peptide, and photorelease is carried out under 365nm ultraviolet light to obtain the adduct.
[0049] S4. Finally, the obtained adducts are detected by LC-MS / MS. The ITCyne-labeled peptides that bind to the L and H probes will generate isotopic labels (isotopic difference 6.0 Da) in LC-MS / MS detection. The probe-labeled peptides are identified by the H / L value during the pFind search process.
[0050] An initial database search was performed using the target modified molecular weight set to the theoretical molecular weight of the thioacrylcysteine adduct (254.08 Da), but no matching peptide profiles were found. Manual retrieval of MS data revealed numerous MS1 peaks with paired isotopic characteristics that had not been identified during the search, suggesting that ITCyne may have unexpected probe-derived modifications (PDM). The data was then reanalyzed using the pChem calculation tool.
[0051] The results are as follows Figure 1As shown in b, a PDM was identified based on the amino acid distribution heatmap of ITCyne-derived modifications obtained from a pChem search. This PDM had a precise mass shift of 220.0957 Da and was primarily located on the N-terminal cysteine residue of the peptide. Based on the elemental composition inferred from the precise modification mass, this unexpected modification likely originated from the initial Cys adduct, in which a hydrogen sulfide molecule (H₂S, 34.0 Da, mass error: 9.0 ppm) was lost.
[0052] Based on this newly discovered PDM, MS data were reanalyzed, identifying 263 isotopically labeled peptides, which could be assigned to 259 adduct cysteine sites. The identification of the 47th cysteine site of the ITCyne adduct site VDAC2 was also conducted.
[0053] Figure 1 c shows the complete annotation of the MS1 spectrum of the ITCyne-labeled cysteine peptide and the MS / MS spectra of the ITCyne-derived modified light and heavy peptides. It can be seen that the dual-charged monoisotopic precursors of the light and heavy peptides were observed at m / z 992.42 (red) and 995.43 (blue), respectively, with a mass error of less than 1.0 ppm, demonstrating that Cys47 of the VDAC2 protein can be modified by ITCyne. Site-specific analysis of the ITCyne adductome using the same method revealed 1282 adductome sites that could be assigned to 991 proteins in three biological replicates.
[0054] Example 3:
[0055] A chemical proteomics workflow for ITC target identification using protein hydrolysis-assisted cyclization (PAC):
[0056] (1) Mechanism Proposal
[0057] The results of cysteine addition sites show that almost all cysteine additions of ITCyne are located at the N-terminus of the identified peptide sequences, and 68.1% of the cysteine addition sites are found to be located on the carboxyl side of lysine (K) or arginine (R) on the protein.
[0058] Figure 2 The stacked bar chart shown in figure a illustrates the frequency of K / R at cleavage sites under different proteases, where T represents trypsin digestion, L represents LysC digestion, and P+T represents tandem digestion based on protein K and trypsin. Therefore, a possible mechanism for the formation of ITCyne-derived protein / peptide adducts is proposed.
[0059] like Figure 2The reaction mechanism shown in the diagram includes the following steps: First, under physiological conditions, ITCyne rapidly forms a thioacyl adduct with the cysteine residues of the protein; second, the peptide bond is hydrolyzed on the K / R carboxyl side adjacent to the initially added cysteine residue; finally, the newly formed primary amine attacks the C=S group through nucleophilic addition to form a tetrahydrothiazole intermediate, which then immediately loses H2S and forms a dihydrothiazole adduct.
[0060] Figure 2 The Venn diagram shows the intersection and differences among the three datasets generated by hydrolysis using different proteases;
[0061] As Figure 2 As shown in c, when LysC was used for in situ digestion of proteomes labeled with ITCyne, the cysteine adducts were mainly found on the carboxyl side of K on the protein (59.1%), rather than R (1.7%).
[0062] (2) Verifying the hypothesis
[0063] The cysteine-containing synthetic peptide (CADSWAGK) was incubated with ITCyne under physiological conditions and analyzed by LC-MS / MS.
[0064] like Figure 2 As shown in d, the time-process stability analysis of the ITCyne-CADSWAGK adducts indicates that the thioyl adduct (m / z 467.681) is rapidly lost within 2 hours at room temperature, while the dihydrothiazole adduct (m / z 450.687) remains stable for at least 24 hours. Figure 2 e demonstrates the ability to generate H2S from isothiocyanates in cell lysates using a lead acetate / lead sulfide assay.
[0065] like Figure 2 As shown in Figure e, when ITCyne was incubated with cell lysates, H2S was observed to form in a protease-dependent manner. These findings suggest that site-specific hydrolysis of peptide bonds is essential for the formation of stable dihydrothiazolium adducts.
[0066] (3) Increase the number of identification sites
[0067] To further enhance the identification of non-K / R adjacent cysteine sites condensed by ITCyne, substrate-broadly specific proteinase K (ProK) was used to generate more novel N-termini of ITCyne-condensed cysteine residues. Specifically, ITCyne-tagged proteomic samples were first subjected to ProK-based restriction proteolysis, followed by trypsin digestion and then further processing as described above. Results showed that this analysis identified 3056 ITCyne-condensed cysteine sites in three biological replicates, attributable to 1967 proteins, covering 64.0% (820) and 63.6% (188) of the cysteine condensations generated by trypsin and LysC digestion, respectively. Only 11.9% and 11.2% of all these cysteines were located on the carboxyl side of K or R, respectively. In three experimental settings, a total of 3572 ITCyne-condensed cysteine sites were identified in HEK293T cells, attributable to 2236 proteins. These results indicate that the PAC reaction can be used for chemical proteomics identification of ITC targets at the site level.
[0068] Example 4:
[0069] Affinity-based chemical proteomics reveals the functional targets of sulforaphane:
[0070] (1) Design and synthesize analogs
[0071] An acetylene analogue of sulforaphane (SFN) was designed and synthesized, hereinafter referred to as SFyne;
[0072] Figure 3 a is a schematic diagram of the chemical structure of sulforaphane and its "clickable" analogue SFyne.
[0073] (2) Target identification
[0074] Direct in situ identification of SFyne-targeted cysteine sites using affinity-based chemical proteomics;
[0075] Figure 3 b describes the in-situ identification process for SFyne targets. Specifically, HEK293T cells are first treated with 100 μM SFyne for 4 h, followed by cell lysis and proteolytic digestion (trypsin and / or proteinase K). Then, the SFyne-modified peptides are bound to the photodegradable Az-UV-Biotin via click chemistry. Next, streptavidin is enriched and photoreleased. Finally, proteomics analysis of HEK293T cells is performed based on LC-MS / MS.
[0076] The results are as follows Figure 3c showed that 255 SFyne-targeting cysteine sites were identified, belonging to 220 proteins. However, some of these cysteine residues are active sites or located within the functional domains of target proteins, potentially leading to SFN-mediated inhibition. Furthermore, GO analysis revealed that the identified target proteins are involved in many SFN-related biological processes and pathways, including cell cycle regulation, the NF-κB pathway, the cytoskeleton, and the heat shock response.
[0077] (3) Functional characterization
[0078] SFyne can cause irreversible arrest of the cell cycle in the G2 / M phase. Among the identified targets of SFyne, the C67 region of PLK1, a key regulator of the mammalian cell cycle, has the highest ratio.
[0079] Figure 3 d is a representative HCDMS / MS spectrum of the SFyne-labeled PLK1-C67 peptide, which can functionally characterize the interaction between SFN and PLK1.
[0080] Image 3e shows the target site PLK1-C67 of SFyne plotted on the crystal structure of human PLK1 protein (PDB#:2OU7) and visualized using Discovery Studio 2.5. Given that C67 is close to the ATP binding site of PLK1, it is initially inferred that SFN may also directly inhibit PLK1 kinase activity. However, ADP-Glo™ kinase analysis showed that SFN only inhibited PLK1 activity in vitro at concentrations above 200 μM, while the positive control staurosporine (Stau) showed effective inhibition of PLK1, with an IC50 < 1 μM. Figure 3 The comparison curves of fSFN and astrosporin on the activity of PLK1 enzyme are shown in the figure.
[0081] Meanwhile, SFN treatment of HEK293T cells for 4 hours led to decreased phosphorylation of BubR1, a substrate of PLK1, indicating a significant reduction in PLK1 activity. Figure 3 As shown in g.
[0082] and Figure 3 Western blot analysis of representative proteins in h showed that increasing SFN concentrations in HEK293T cells (0, 1, 10, 20, 40, 60, and 80 μM, 4 h each) led to decreased expression of endogenous PLK1, consistent with shRNA-mediated knockout. Furthermore:
[0083] Figure 3Representative Western blot analysis showed that treatment of HEK293T cells with the proteasome inhibitor MG132 attenuated SFN-induced PLK1 degradation. The experimental group cells were pre-incubated with DMSO or MG132 (5 μM, 24 h), followed by treatment with 100 μM SFN for 4 h. The control group received the corresponding concentration of DMSO. The results indicated that the SFN-mediated decrease in PLK1 levels could be eliminated by the proteasome inhibitor MG132. This functional characterization suggests that the loss of PLK1 kinase activity may be attributed to SFN-mediated protein degradation rather than inhibition.
[0084] (4) Study the impact of mutations
[0085] To investigate the effect of the C67S mutation on the interaction between SFN and PLK1, HEK293T cells were transiently transfected with overexpression of wild-type (WT) or C67S mutant PLK1 protein.
[0086] Figure 3 Western blot analysis of representative proteins showed that the C67S mutation weakened the SFN-induced degradation of PLK1. HEK293T cells expressing wild-type and C67S mutant PLK1 were treated with different concentrations of SFN (0, 25, 50, 75, 100 μM), and the expression of PLK1 protein was detected by Western blot after 4 h.
[0087] Figure 3 k shows the survival rate of HEK293T cells expressing PLK1 wild-type and C67S mutant cells as detected by MTT assay. The figure shows representative data from three replicate experiments, and the statistical significance of error bars and standard deviations was determined by two-tailed t-test.
[0088] The results are as follows Figure 3 j and Figure 3 k showed that the C67S mutation significantly attenuated SFN-induced PLK1 degradation and cytotoxic effects. In summary, these results indicate that SFN exerts its cytotoxic effects at least partially through cysteine-mediated covalent modification and PLK1 degradation, revealing PLK1-C67 as a functional target of sulforaphane.
[0089] The foregoing has only described certain exemplary embodiments of the present invention by way of illustration. Undoubtedly, those skilled in the art can modify the described embodiments in various ways without departing from the spirit and scope of the present invention. Therefore, the foregoing drawings and descriptions are illustrative in nature and should not be construed as limiting the scope of protection of the claims of the present invention.
Claims
1. A method for identifying isothiocyanate targets based on protein hydrolysis-assisted cyclization reaction, characterized in that, Specifically, the following steps are included: S1. Incubate the isothiocyanate-containing chemical probe with the biological sample, and then react the isothiocyanate-containing chemical probe with the protein cysteine residues in the biological sample to obtain isothiocyanate-labeled protein. S2. The isothiocyanate-labeled protein is digested by protease to obtain a short peptide containing an N-terminus. Then, a proteolytic cyclization reaction of the short peptide containing an N-terminus is triggered to obtain an isothiocyanate-modified peptide. S3. The isothiocyanate-modified peptide is coupled with a biotinylated reagent through a click chemical reaction, and then the coupling product is captured by streptavidin and photodegraded by ultraviolet light to finally obtain a cysteine adduct. S4. The cysteine adduct was analyzed by liquid chromatography-tandem mass spectrometry, and the analysis results were then analyzed using computational tools to identify the cysteine adduct sites and their corresponding protein targets.
2. The method for isothiocyanate target identification based on protein hydrolysis-assisted cyclization reaction according to claim 1, characterized in that, The isothiocyanate-containing chemical probe in S1 is an isothiocyanate analog containing an azide group or an alkyne group.
3. The method for isothiocyanate target identification based on protein hydrolysis-assisted cyclization reaction according to claim 2, characterized in that, The isothiocyanate analogue is propargyl isothiocyanate or an alkyne derivative of sulforaphane.
4. The method for isothiocyanate target identification based on protein hydrolysis-assisted cyclization reaction according to claim 1, characterized in that, The biological sample in S1 is at least one of cell lysate, tissue extract, or in vitro cultured intact cells.
5. The method for isothiocyanate target identification based on protein hydrolysis-assisted cyclization reaction according to claim 1, characterized in that, The protease in S2 is at least one of trypsin, LysC, and proteinase K.
6. The method for isothiocyanate target identification based on protein hydrolysis-assisted cyclization reaction according to claim 5, characterized in that, The protease hydrolysis is a tandem digestion, including the following steps: A1. First, the isothiocyanate-labeled protein was alkylated with iodoacetamide, and then restricted hydrolysis was performed using proteinase K to obtain a long peptide containing isothiocyanate adduct. A2. Subsequently, a second digestion was performed using trypsin or LysC to cleave the long peptide containing the isothiocyanate adduct, yielding a short peptide with an N-terminus.
7. The method for isothiocyanate target identification based on protein hydrolysis-assisted cyclization reaction according to claim 1, characterized in that, The biotinylating agent in S3 is a light azide-UV cleavable biotin and an eclipsed nitrogen-UV cleavable biotin.
8. The method for isothiocyanate target identification based on protein hydrolysis-assisted cyclization reaction according to claim 1, characterized in that, The calculation tool in S4 is a modification site analysis software based on mass spectrometry data, used to identify the precise mass shift of cysteine adducts and their corresponding peptide sequences.