Quantitative chemical proteomic method for lipoic-modification sites

Abstract

This application relates to the field of chemical proteomics, and particularly to a quantitative chemical proteomics method for thiocylation modification sites. It employs aldehyde and alkyne-containing probes to chemically selectively label the proteome of biological samples with thiocylation modification. Quantitative chemical proteomics techniques are then used to quantitatively analyze the chemically labeled thiocylation modification sites. This method enables the identification and quantification of all thiocylation modification sites in biological samples, providing an analytical tool for studying the effects of thiocylation modification on protein structure and function. It facilitates research into the molecular mechanisms of thiocylation modification in related diseases, achieving high coverage, high reproducibility, and high accuracy in target screening, and providing corresponding technical support for subsequent drug development.

Description

Technical Field

[0001] This application relates to the field of chemical proteomics, and more particularly to quantitative chemical proteomics methods for thioctylation modification sites. Background Technology

[0002] Lipoacetylation is a posttranslational modification (PTM) that covalently links lipoic acid to lysine residues in proteins via an amide bond. It serves as the active site for several protein complexes in core metabolic pathways (such as the pyruvate dehydrogenase complex, ketoglutarate dehydrogenase complex, and branched-chain ketoate dehydrogenase complex), playing a crucial catalytic role as a key cofactor. The pyruvate dehydrogenase complex (PDH) is a multifunctional enzyme complex in which the two sulfur atoms in the lipoic acid of the dihydrolipoamide transacetylase (E2) subunit interconvert between the disulfide bond and the thiol group through a redox cycle, completing catalytic processes such as acyl transfer and electron transport to catalyze the conversion of pyruvate to acetyl-CoA, thus linking glycolysis to the tricarboxylic acid cycle. Dysregulation of PDH can lead to metabolic disorders, cancer, and Alzheimer's disease in humans. Therefore, advancing the understanding of the regulation of lipoacetylation is crucial for investigating the underlying molecular mechanisms of these diseases.

[0003] Liposylation modification is highly conserved evolutionarily, with only 3 and 5 liposylated proteins (5 and 6 liposylation sites) found in the E. coli and mammalian proteomes, respectively. Early work mainly used structural and biochemical methods, such as Western blotting, NMR mapping, and amino acid sequencing, to study the liposylation modification of individual proteins.

[0004] Recent mass spectrometry-based methods have provided a means to study the thiocylation modification status of specific lysine residues in different cell types, tissues, and biological environments. In 2019, Kajimura's group integrated immunoprecipitation with anti-thiocylation antibodies and mass spectrometry to identify thiocylated proteins in brown adipose tissue in the scapula of mice. However, due to the limited binding of the antibodies, their affinity was insufficient to persist under strict denaturing and washing conditions, preventing the identification of all thiocylated proteins. In 2021, Xing Chen's group integrated iodoacetamide-assisted cyclooctyne linkage (iLCL) labeling and mass spectrometry to identify all thiocylated proteins in E. coli and mouse cell lines. However, this technique has not been developed into a method capable of identifying thiocylation modification sites. In 2014, Cristea's group used targeted mass spectrometry to selectively detect and quantify the level of thiocylation modification in different biological samples, thereby enabling the in vitro and in vivo measurement of the activity of the dethiocylase SIRT4. Targeted protein mass spectrometry analysis has been successfully used to quantify lipoylation modification sites in the pyruvate dehydrogenase complex in humans. However, current techniques are unable to quantify all lipoylation modifications at the proteomic level. Therefore, a specific quantitative method for globally analyzing lipoylation modification sites in the proteome remains lacking. Summary of the Invention

[0005] To specifically quantify all thiocylation modification sites in the proteome, this application provides a quantitative chemical proteomics method for thiocylation modification sites. This method involves chemically selectively labeling thiocylation modifications in the proteome of biological samples using probes containing aldehyde and alkyne groups; then, quantitative chemical proteomics techniques are used to quantitatively analyze the chemically labeled sites. This enables the identification and quantification of all thiocylation modification sites in biological samples, providing an analytical tool for studying the effects of thiocylation modification on protein structure and function, and facilitating research into the molecular mechanisms of thiocylation modification in related diseases.

[0006] In one aspect, this application provides a quantitative chemical proteomics method for thiocylation modification sites, including:

[0007] Chemically selective labeling of the proteome of biological samples was performed by thiocrystalization using probes containing aldehyde and alkyne groups; quantitative analysis of the chemically selectively labeled sites was then conducted using quantitative chemical proteomics techniques.

[0008] In one embodiment, the biological sample includes: Escherichia coli, yeast cells, human cell lines, human tissue samples, human organs, mouse cell lines, mouse tissue samples, mouse organs, Drosophila cell lines, zebrafish cell lines, or nematode cell lines.

[0009] In one embodiment, the biological sample further includes: animal body fluids (such as urine, blood, saliva, bile, gastric juice, lymph, and other secretions of the organism), hair, muscles, and some tissues and organs (such as thymus, pancreas, liver, lungs, brain, stomach, kidneys, etc.) as well as various microorganisms.

[0010] In one embodiment, the structure of the probe containing aldehyde and alkyne groups is as follows:

[0011]

[0012] n = 1-9, where n is an integer.

[0013] In one embodiment, the probe containing aldehyde and alkynyl groups is a butyraldehyde probe (BAP), with the following structure:

[0014]

[0015] In one embodiment, the steps for synthesizing the alkynylbutyraldehyde probe include: sequentially adding dichloromethane and Dess-Martin Periodinane reagent, stirring in an ice bath to allow it to cool completely; adding 5-hexyn-1-ol, followed by stirring, cooling, ultrafiltration, rotary evaporation, and purification by column chromatography using a 5:1 cyclohexane:diethyl ether solution to obtain the alkynylbutyraldehyde probe.

[0016] In one embodiment, the steps for synthesizing the alkynylbutyraldehyde probe include: adding 10-30 mL of dichloromethane to a round-bottom flask, followed by 1.2-3.2 g of Dess-Martin Periodinane reagent, stirring in an ice bath for 5-15 min to allow it to cool completely. Then, adding 0.3-0.7 mL of 5-hexyn-1-ol using a constant-pressure syringe. Stirring overnight. Cooling on ice the next day. Filtering out excess Dess-Martin Periodinane using an ultrafiltration sintered glass funnel lined with silica gel, followed by rotary evaporation at 2-8°C to remove excess solvent, and then purifying by column chromatography with a 5:1 cyclohexane:diethyl ether solution to obtain the final alkynylbutyraldehyde probe.

[0017] In one embodiment, the quantitative analysis includes biological sample pretreatment and mass spectrometry analysis. The biological sample pretreatment steps include: click chemistry reaction of chemically selectively labeled proteins, protein precipitation, streptavidin bead enrichment and alkylation, enzyme digestion, dimethylation quantification, and acid digestion.

[0018] In one embodiment, the click chemistry step includes: resolving the protein precipitate in SDS-PBS (PBS solution containing SDS), adding an acid-cleaved enrichment tag (Dialkoxydiphenylsilane, abbreviated as DADPS), CuSO4, TBTA tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine and TCEP (tris(2-carboxyethyl)phosphine) and performing a click chemistry reaction at room temperature.

[0019] In one embodiment, the protein precipitation step includes: adding methanol, chloroform and deionized water to the reaction system, centrifuging to precipitate the protein at room temperature, and washing the precipitate twice with cold methanol.

[0020] In one embodiment, the streptavidin bead enrichment step includes: resolving the protein precipitate in SDS-containing PBS, adding it to the streptavidin beads, resulting in a final system of 0.2% SDS-containing PBS, enriching at 29°C, followed by centrifugation and discarding the supernatant. The streptavidin beads are then washed three times with PBS, followed by three washes with deionized water to remove SDS and non-specifically adsorbed proteins.

[0021] In one embodiment, the alkylation step includes: transferring streptavidin beads into a screw-top Eppendorf tube and adding PBS containing urea. Then, DTT (dithiothreitol) is added and the reaction is carried out at 37°C. IAA (iodoacetamide) is then added and the reaction is carried out at 35°C, ensuring that all disulfide bonds opened by DTT react with IAA to prevent re-formation of disulfide bonds.

[0022] It should also be noted that any probe involved in this application that has a functional group capable of reacting with lipoic acid and a group for copper-catalyzed click chemistry reactions, and whose carbon number is within a certain range (including benzene rings), falls within the scope of the probe synthesis concept of this application and is protected within the scope of this application.

[0023] In one implementation, when the biological sample is *E. coli*, the pretreatment enzyme digestion steps for quantitative analysis of ODO2 protein (dihydrolipoyllysine succinyltransferase (Escherichia coli), abbreviated as ODO2) and GCSH protein (glycine cleavage system H protein, abbreviated as GCSH) sites include:

[0024] Enzymatic digestion was performed using V8 protease (Glu-C protease) dissolved in ammonium bicarbonate solution.

[0025] In one implementation, when the biological sample is *E. coli*, the pretreatment enzymatic digestion steps for quantitative analysis of three sites of the ODP2 protein (Dihydrolipoyllysine acetyltransferase (Escherichia coli), abbreviated as ODP2) in *E. coli* include:

[0026] Enzymatic digestion was performed using trypsin dissolved in phosphate buffer solution. After the acid digestion step, enzymatic digestion was performed again using V8 protease dissolved in ammonium bicarbonate solution.

[0027] In one implementation, the pretreatment step for probe-modified E. coli includes:

[0028] Frozen *E. coli* were obtained and added to an SDS-based ABS solution, followed by sonication to lyse the cells. The ABS solution was prepared by mixing 20.55 mL of 0.2 M disodium hydrogen phosphate and 79.45 mL of 0.1 M citric acid to obtain 100 mL of pH 3 acidic buffer solution (ABS). After centrifugation, the supernatant was collected and the protein concentration was adjusted. The supernatant protein was divided into three groups: two groups were BAP groups, which were reacted with TCEP followed by the addition of a BAP probe; the other group was DMSO groups, which were reacted with TCEP followed by the addition of an equal amount of DMSO as a control. Precipitation, click chemistry reaction, streptavidin bead enrichment and alkylation, and enzyme digestion were performed sequentially.

[0029] If the thioctylation modification of ODO2 and GCSH in Escherichia coli is to be quantified, the enzyme digestion steps are as follows: V8 protease dissolved in ammonium bicarbonate solution is added to the system, and after complete enzyme digestion, the system is washed with ammonium bicarbonate and deionized water in sequence; then, the dimethylation quantification step and the acid digestion step are performed.

[0030] To quantify the three lipoylation modifications of ODP2 in *E. coli*, the enzymatic digestion steps are as follows: Add a solution containing urea, PBS, and CaCl2, along with trypsin, to the system. After complete digestion, wash sequentially with ammonium bicarbonate and deionized water. Then perform a dimethylation quantification step and an acid digestion step. Perform a second digestion: add V8 protease dissolved in ammonium bicarbonate solution for digestion.

[0031] Dimethylation quantification procedure: When there are 3 samples, the three groups of protein samples are labeled as light-labeled, medium-labeled, and heavy-labeled; HCHO and NaBH3CN are added to the light-labeled samples; DCDO and NaBH3CN are added to the medium-labeled samples; and D2CO3 is added to the heavy-labeled samples. 13 CDO and NaBD3CN were used to wash the three groups of samples sequentially with TEAB (triethylammonium bicarbonate) and deionized water, respectively. Streptomycin beads were mixed in a 1:1:1 ratio and then washed.

[0032] When the sample size is 2, the three protein samples are labeled as lightly labeled and heavily labeled; HCHO and NaBH3CN are added to the lightly labeled samples; D is added to the heavily labeled samples. 13 CDO and NaBH3CN were used to wash the three groups of samples sequentially with TEAB and deionized water. Streptomycin beads were mixed at a 1:1 ratio and then washed.

[0033] Acid cutting steps: Add formic acid-water for acid cutting. After complete acid cutting, combine the supernatants, evaporate to dryness, and store for mass spectrometry analysis.

[0034] In one implementation, when the biological sample is a human cell line, the pretreatment enzyme digestion step for quantitative analysis of all sites in the human cell line includes:

[0035] Trypsin and V8 protease dissolved in phosphate buffer solution were added simultaneously for enzymatic digestion.

[0036] In one implementation, the pretreatment steps for quantitative analysis of all thioctylation modification sites in human cell lines include:

[0037] Cryopreserved human cell lines were obtained and lysed in SDS-ABS solution by sonication. The cells were centrifuged, and the supernatant was collected and its protein concentration adjusted. TCEP and BAP probes were added sequentially for reaction. After complete reaction, precipitation, click chemistry, streptavidin bead enrichment, alkylation, and enzyme digestion were performed sequentially. Urea-containing PBS solution and trypsin were added to the system for enzyme digestion, followed by washing with PBS and deionized water. Urea-containing ammonium bicarbonate solution was added to the system, followed by V8 protease digestion, followed by washing with ammonium bicarbonate and deionized water. Acid digestion was performed using 2% formic acid-water mass spectrometry, with the acid digestion enrichment tag CY58. The supernatants from the acid digestion were combined, evaporated to dryness, and stored for mass spectrometry analysis.

[0038] In one implementation, when the biological sample is a human cell line, the CY58 acid-cutterable tag is used for cutting during the acid cutting step. The CY58 tag has the following structural formula:

[0039]

[0040] In one implementation, CY58 can also be replaced by an acidic cleavable tag with the following structure, which represents the application of an "acidic cleavable tag with an additional positively charged group" in thiocrylation modification, as shown below:

[0041]

[0042] Where X represents a positively charged functional group, including but not limited to nitrogen atoms in primary, secondary, and tertiary fatty amines, imine nitrogen atoms in nitrogen-nitrogen disubstituted amidine groups, imine nitrogen atoms in tetranitrogen substituted guanidine groups, amidine nitrogen atoms containing at least one unsubstituted hydrogen atom, and guanidine nitrogen atoms containing at least one unsubstituted hydrogen atom.

[0043] In one implementation scheme, the synthesis steps of CY58 are as follows:

[0044]

[0045] In one aspect, the compounds CY56, CY57, and CY58 mentioned in the quantitative chemical proteomics method for the thioctylated modification sites have the following structural formulas:

[0046]

[0047] In one embodiment, when the biological sample is *E. coli*, the quantitative chemical proteomics method for the thioctylation modification sites can identify and quantify five thioctylation modification sites in the *E. coli* proteome; or,

[0048] When the biological sample is a human proteome sample, the quantitative chemical proteomics method for the thioctylation modification sites can identify and quantify six thioctylation modification sites in the human cell line proteome.

[0049] It should be noted that the temperature conditions involved in this application (such as -80℃, -20℃, 4℃, 20℃, etc.) are all range temperature values. As long as the temperature can achieve the experimental purpose involved in this application, such as freezing or thawing, any such range temperature value falls within the protection scope of this application.

[0050] The centrifugation conditions (such as rotation speed, time, temperature, etc.) involved in this application, as long as they can achieve the centrifugation purpose involved in this application, are simply variations of the centrifugation conditions involved in this application and all fall within the protection scope of this application.

[0051] In one embodiment, the washing reagent involved in this application is not limited to PBS or deionized water, but may also be other washing reagents.

[0052] In one implementation, the pretreated biological sample is subjected to mass spectrometry analysis using LC-MS / MS, and the LC-MS / MS data analysis is performed using ProLuCID, with fixed modifications on cysteine ​​residues and variable modifications on methionine residues.

[0053] In one implementation, the pretreated biological samples are subjected to mass spectrometry analysis using LC-MS / MS. The LC-MS / MS data analysis is performed using ProLuCID, with a fixed modification of +57.0215 Da on cysteine ​​residues and a variable modification of +15.9949 Da on methionine residues.

[0054] When identifying variable modifications of thiocyl acylation in bovine serum albumin (BSA) and Escherichia coli, the lysine residue increased by 411.20132 Da. When identifying variable modifications of thiocyl acylation in human cell lines, the lysine residue increased by 511.26508 Da. Attached Figure Description

[0055] To more clearly illustrate the technical solution of this application, the drawings used in the embodiments will be briefly introduced below. Obviously, for those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0056] Figure 1To test the reactivity of BAP in a small molecule reaction model, (a) lipoic acid (LA) reacted with BAP in an acidic buffer system at pH 3 to form the linker product LA-BAP. TCEP was added before the reaction to reduce the disulfide bonds in LA; (b) LC-MS analysis of the reaction between LA and BAP was performed, measuring the absorbance of LA and LA-BAP at 270 nm; (c) LA-BAP mass spectrometry showed that the theoretical molecular weight of the product was 285.1; retention time.

[0057] Figure 2 This section describes the chemoselective labeling of purified proteins using alkynylbutyraldehyde (BAP) probes for thiocylation modification; (a) the reaction protocol for BAP capture of thiocylation modification; (b) the experimental procedure for BAP labeling of thiocylated bovine serum albumin (BSA). BSA reacts with NHS-LA to form thiocylated BSA, which is then labeled with BAP and analyzed by in-gel fluorescence imaging or LC-MS / MS; (c) fluorescence imaging and Coomassie blue staining of BAP-labeled BSA after gel electrophoresis; (d) showing one of the classic MS / MS spectra: MS / MS spectrum of LSQK*FPL, with corresponding b and y ions labeled; gel fluorescence imaging (Rho), Coomassie Brilliant Blue (CBB), relative intensity, and mass-to-nucleus ratio (m / z).

[0058] Figure 3 To identify lipoylation modification sites in the E. coli proteome; (a) Schematic diagram of identification of lipoylation modification sites using a mass spectrometry-based tandem orthogonal proteolysis strategy; (b) Comparison of results of identification of lipoylation modification in the E. coli proteome using trypsin or Glu-C, * indicates that the three lipoylation modification sites in ODP2 produce the same lipoylation modified peptide after Glu-C digestion; (c) MS / MS spectra of the three lipoylation modified peptides in E. coli after Glu-C digestion; the corresponding b and y ions are labeled.

[0059] Figure 4To assess the quantitative accuracy of thiocylation modification using a developed mass spectrometry method via dimethylation; (a) a flowchart of mass spectrometry-based thiocylation site analysis combined with dimethyl labeling; (b) primary chromatograms and quantitative ratios of thiocylated peptides extracted from GCSH, ODO2, and ODP2, with the three thiocylated peptides in ODP2 exhibiting identical sequences after Glu-C digestion; (c) quantitative accuracy of thiocylation modification sites in the E. coli proteome from three replicate experiments; (d) the quantitative ratios of extracted primary chromatograms and downstream characteristic peptides of the three thiocylation modification sites in ODP2, which can be used as "surrogate peptides" to quantify the thiocylation modification level at each site in ODP2; in (b) and (d), protein names and peptide sequences are shown at the top, with quantitative ratios (R... L / M and R H / M (e) The accuracy of quantification of thioctylation sites of ODP2 in the E. coli proteome from three experiments is shown below.

[0060] Figure 5 Quantitative analysis of three lipoylation modification sites in ODP2; (a) Using a tandem enzyme digestion process, based on the downstream characteristic sequence of ODP2, the workflow for distinguishing and quantifying three evolutionarily conserved lipoylation modification sites in ODP2 was employed. After trypsin digestion, the ε-amino group of the N-terminus and lysine residues of the peptide was isotopically labeled by reductive dimethylation. Subsequent Glu-C digestion released the characteristic “alternative peptides,” and quantification was performed using the peptides downstream of each lipoylation modification site in ODP2; (b) Quantification was performed by comparing wild-type and ODP2 mutant strains. (c) Dimethylation quantification of thiocylation modifications between ODP2 sites was performed to explore the regulatory workflow of the three thiocylation modification sites in ODP2. Mutant strains included ODP2 knockout (ΔODP2) strains and strains expressing exogenous ODP2 with single mutants (K41R, K144R, K245R) or their paired combinations (double mutants); (d) Quantification of the level of each thiocylation modification at each of the three sites in ODP2 after single or double mutations of ODP2 knockout or thiocylation of lysine was performed in three biological replicates.

[0061] Figure 6Quantification of lipoylation modification following genetic perturbation of lipoyl synthases in *E. coli*; (a) The figure shows the biosynthetic pathway of protein lipoylation modification in *E. coli*, including de novo synthesis and direct synthesis pathways, with key lipoyl synthases highlighted in color, including LipA, LipB, and LplA; (b) Quantification of the levels of five known lipoylation modification sites after knocking out one or two different lipoyl synthases in conventional LB medium; (c) Quantification of the levels of five known lipoylation modification sites after knocking out a single different lipoyl synthase in conditioned medium with glucose as the sole carbon source; In (b) and (c), the color intensity of the heatmap corresponds to the quantitative ratio between knockout and wild-type strains.

[0062] Figure 7 Quantitative analysis of lipoylation modification sites in the human proteome; (a) Sequences of all six lipoylation modified peptides and comparison of identification results using DADPS and CY58; (b) Structures of DADPS and CY58; (c) Workflow for quantifying changes in lipoylation modification levels in human cancer cell lines after knockout of lipoyl synthase LIAS; (d) Quantification of the levels of six known lipoylation modification sites in human cells after LIAS knockdown by siRNA; (e) Detection of endogenous GCSH mass transfer induced by protein lipoylation modification using the azide-2k-PEG mass tag, with dashed and solid arrows representing unmodified and lipoylated proteins, respectively; (f) Quantification of lipoylation modification chemometry of GCSH in K562 and HepG2 cells based on the presence or absence of GCSH mass transfer using Western blotting.

[0063] Figure 8 Chemical characterization of compound CY56, (a) 1 (b) H NMR spectrum; 13 (c) C NMR spectrum; (c) Fourier transform high-resolution mass spectrometry.

[0064] Figure 9 Chemical characterization of compound CY57, (a) 1 (b) H NMR spectrum; 13 (c) C NMR spectrum; (c) Fourier transform high-resolution mass spectrometry.

[0065] Figure 10 Chemical characterization of compound CY58, (a) 1 (b) H NMR spectrum; 13 (c) C NMR spectrum; (c) Fourier transform high-resolution mass spectrometry. Detailed Implementation

[0066] The embodiments will now be described in detail, examples of which are illustrated in the accompanying drawings. When the following description relates to the drawings, unless otherwise indicated, the same numbers in different drawings represent the same or similar elements. The embodiments described below do not represent all embodiments consistent with this application. They are merely examples of systems and methods consistent with some aspects of this application as detailed in the claims.

[0067] Example 1: Development of a probe for chemically selective labeling of thiocyl-modified alkynylbutyraldehyde

[0068] This application first designed and synthesized an alkynylbutyraldehyde probe, which contains an aldehyde group that reacts with lipoic acid (LA) and a bioorthogonal alkyne group for copper-catalyzed click chemistry reactions (see [link to application]). Figure 1 (a) First, the applicant evaluated the reactivity of BAP with LA under acidic conditions in a small molecule model. LC-MS analysis detected the expected product LA-BAP (see [reference]). Figure 1 (b) has a corresponding molecular weight of 286 Da (see [reference]). Figure 1 (c)

[0069] The synthesis steps of BAP are as follows:

[0070] Add 20 mL of dichloromethane to a 50 mL round-bottom flask, followed by 2.2 g of Dess-Martin Periodinane reagent. Stir in an ice bath for 10 min to allow it to cool completely. Then, add 0.56 mL of 5-hexyn-1-ol using a constant-pressure syringe. Stir overnight. Cool on ice the next day. Filter excess Dess-Martin Periodinane using an ultrafiltration sintered glass funnel covered with silica gel. Rotary evaporate at 4 °C to remove excess solvent. Purify by column chromatography using a 5:1 cyclohexane:diethyl ether solution. The final amount of BAP obtained was 350 mg, with a theoretical yield of 92%. 1H NMR (400MHz, Chloroform-d) δ9.81 (s, 1H), 2.64-2.60 (m, 2H), 2.30-2.26 (m, 2H), 1.99 (t, J = 2.6Hz, 1H), 1.86 (p, J = 7.0Hz, 2H).

[0071] Example 2: Chemically selective labeling of purified protein by thiocylation modification using an alkynylbutyraldehyde probe.

[0072] This application's embodiments tested the labeling of lipoic acid acylation modifications on pure proteins using BAP. This was achieved by synthesizing N-hydroxysuccinimide ester of lipoic acid (NHS-LA) (see...). Figure 2(a) and incubated with bovine serum albumin (BSA) to generate thioctylated modified BSA (LA-BSA), which was then labeled with a gel fluorescent marker (see [link]). Figure 2 (b) In the presence of 2 mM BAP, fluorescence was detected only in the LA-BSA sample, but not in the negative control (see [reference]). Figure 2 (c)

[0073] To further confirm the reaction between BAP and LAP-BSA, the applicant prepared a mass spectrometry sample and analyzed it by liquid chromatography-tandem MS (LC-MS / MS), successfully identifying all thiocylation modification sites (see [link]). Figure 2 (d). In summary, these results demonstrate that BAP can chemically selectively label small molecules and purify thiocylation modifications in proteins.

[0074] The synthesis steps of NHS-LA are as follows:

[0075] Lipoic acid (206 mg, 1 mmol) and N-hydroxysuccinimide (115 mg, 1 mmol) were dissolved in 25 mL of THF. N'-diisopropylcarbodiimide (0.15 mL, 1 mmol) was diluted with 5 mL of THF and then added dropwise to the reaction mixture. The reaction was stirred overnight at room temperature. Diisopropylurea precipitated from the solution during the reaction and was removed by filtration. The filtrate was concentrated under vacuum using a rotary evaporator to obtain a yellow solid weighing 5.5 g, yielding a final theoretical yield of 93%. (400MHz, Chloroform-d) δ3.60-3.57(m,1H),3.24-3.07(m,2H),2.84(s,4H),2.63(t,J= 7.4Hz,2H),2.51-2.44(m,1H),1.97-1.89(m,1H),1.85-1.67(m,4H),1.61-1.54(m,2H).

[0076] Example 3: Identification of lipoylation modification sites in the E. coli proteome

[0077] This application further utilizes BAP to identify lipoylation modification sites in the E. coli proteome. Five known lipoylation modification sites are found in three lipoylation modification proteins in E. coli, including three in dihydrolipoyllysine acetyltransferase (Escherichia coli, ODP2), one in dihydrolipoyllysine succinyltransferase (Escherichia coli, ODO2), and one in glycine cleavage system H protein (GCSH). Tandem orthogonal proteolysis-activity-based protein profiling (TOP-ABPP) is a technique for analyzing active proteins. The TOP-ABPP technique can be referenced in the following two articles: Reference 1: Speers, AE & Cravatt, BFA. Tandem orthogonal proteolysis strategy for high-content chemical proteomics. J Am Chem Soc 127, 10018-10019 (2005). Reference 2: Weerapana, E., Speers, AE & Cravatt, BFA. Tandem orthogonal proteolysis-activity-based protein profiling (TOP-ABPP) - a general method for mapping sites of probe modification in proteomes. Nat Protoc 2, 1414-1425 (2007).

[0078] TOP-ABPP employs a standard chemical proteomics workflow (see [link]). Figure 3 In section a), the sequences of these lipoylated peptides could not be identified when digested with trypsin, a commonly used proteomics enzyme. This is mainly due to the following technical difficulties: ① The lipoylated peptides produced by trypsin digestion are too long to be identified by conventional LC-MS / MS. ② The lipoylation sites have multiple aspartic (D) and glutamic (E) residues flanking the lysine residue, which further reduces the mass spectrometry performance of lipoylated peptides in positive ion mode.

[0079] To overcome this technical challenge, the applicant utilized the property of Glu-C to hydrolyze the peptide bonds at the D and E carboxyl ends, further replacing Trypsin with Glu-C in a standard chemical proteomics workflow. This ultimately led to the successful identification of all thiocyl-modified peptides in three substrate proteins from *E. coli* (see [link to documentation]). Figure 3 (b) This result yielded the MS / MS spectrum (see [reference]). Figure 3 Support for (c).

[0080] Example 4: Establishment of a site-specific quantitative protocol for lipoylation in the E. coli proteome

[0081] After establishing a workflow for identifying known thioctylation sites in *E. coli*, the quantitative accuracy of this workflow was evaluated in the embodiments of this application. Reductive dimethylation was selected as the quantitative labeling method. *E. coli* lysates were divided into three equal portions, two labeled with 20 mM BAP and one treated with DMSO (see [link to documentation]). Figure 4 (a) Following the TOP-ABPP workflow, the glut-C digested lipoyl peptides of the three samples were isotopically labeled with light, medium, and heavy dimethylating agents (for samples treated with DMSO, BAP, and BAP, respectively). The dimethylated peptides were analyzed by LC-MS / MS. Quantitative results showed that the average neutral / heavy labeling ratio was close to 1 for all lipoyl peptides from the three lipoylated proteins, confirming the accuracy of the quantification. The light-labeled group showed almost no detectable signal, confirming the specificity of the method. (See [link to documentation]) Figure 4 b and Figure 4 (c)

[0082] Unlike the lipoylated peptides in GCSH and ODO2, which have unique sequences, the three lipoylation sites in ODP2 have identical sequences after Glu-C cleavage. To distinguish these three lipoylation sites in ODP2, this application modifies the process by using the downstream characteristic sequences of the lipoylation for quantification.

[0083] More specifically, some improvements were made: after BAP labeling and copper-catalyzed click reaction with DADPS, the enriched ODP2 was first cleaved with Trypsin, then with Glu-C tandem cleavage, thereby releasing the characteristic sequence downstream of the C-terminus of the three thiocylation sites (see [link to relevant documentation]). Figure 5 (a) Then, these "substituted" peptides were dimethylated for quantification. The results showed that the three thiocylation modification sites in ODP2 could be successfully distinguished and accurately quantified using the "substituted" peptides, with an average positive / negative ratio close to 1, confirming the accuracy of the quantification (see [reference]). Figure 4 d and Figure 4(e). Ultimately, all five known thiocylation modification sites in E. coli can be distinguished, identified, and quantified using this technique.

[0084] Quantitative steps for the lipoylation modification of ODO2 and GCSH in Escherichia coli:

[0085] K12 E. coli stored at -80℃ were removed and added to an ABS solution containing 1.2% SDS, followed by sonication to lyse the cells. The ABS solution was prepared by mixing 20.55 mL of 0.2 M Na₂HPO₄ and 79.45 mL of 0.1 M citric acid to obtain 100 mL of acid buffer solution (ABS) with a pH of 3. The solution was then centrifuged at 20000 g for 1 h at 20℃.

[0086] Probe reaction: There were three groups of protein samples: two groups of protein samples (BAP group) were reacted with 10 mM TCEP for 30 min, followed by the addition of 20 mM BAP probe. The reaction was carried out at 37℃ for 12 h. The other group of protein samples (DMSO group) was reacted with 10 mM TCEP for 30 min, followed by the addition of an equal volume of DMSO as a control. Subsequent precipitation, click chemistry reaction, streptavidin bead enrichment, and alkylation steps were performed. Enzyme digestion step: 100 μL of 100 mM ammonium bicarbonate solution containing 1 M Urea was added to the system, followed by 4 μg V8 protease. Enzyme digestion was carried out at 37℃ for 12 h. After 12 h, the sample was washed three times with 1 mL of 100 mM ammonium bicarbonate, and then three times with 1 mL of deionized water.

[0087] Dimethylation quantification steps: The three groups of protein samples described in this application embodiment were labeled as light-labeled, medium-labeled, and heavy-labeled. In the light-labeled samples, 8 μL of 4% light-labeled formaldehyde (HCHO) and 8 μL of 0.6M NaBH3CN were added to every 100 μL of TEAB; in the medium-labeled samples, 8 μL of 4% medium-labeled formaldehyde (DCDO) and 8 μL of 0.6M NaBH3CN were added to every 100 μL of TEAB; in the heavy-labeled samples, 8 μL of 4% heavy-labeled formaldehyde (DCDO) was added to every 100 μL of TEAB. 13 CDO) and 8 μL of 0.6M NaBD3CN were reacted at 25℃ for 2 h. After 2 h, the three groups of samples were washed twice with TEAB and then twice with deionized water. Streptomycin beads were mixed at a ratio of 1:1:1 and then washed twice with deionized water.

[0088] Acid digestion: Add 200 μL of 2% formic acid-water mixture and perform acid digestion for 1 h, repeating twice. Combine the supernatants from the acid digestion, evaporate to dryness, and store the sample at -20℃ for mass spectrometry analysis.

[0089] All subsequent precipitation, click chemical reaction, streptavidin bead enrichment and alkylation steps appearing in the embodiments of this application are as follows:

[0090] Click chemistry reaction procedure: The protein precipitate was reconstituted into a 0.4% SDS-PBS solution. Then, 100 μM acid-cleaved enrichment tag DADPS, 1 mM CuSO4, 100 μM TBTA, and 1 mM TCEP were added, and the click chemistry reaction was carried out at room temperature for 1 h.

[0091] Protein precipitation steps: Add 400 μL methanol, 100 μL chloroform and 300 μL deionized water to the reaction system, centrifuge at 10000g at room temperature for 10 min to precipitate the protein, and wash the precipitate twice with cold methanol at -80℃.

[0092] Streptavidin bead enrichment procedure: The protein precipitate was reconstituted in 1 mL of 1.2% SDS-PBS, and then added to 5 mL of PBS containing streptavidin beads, making the final system 0.2% SDS-PBS. Enrichment was carried out at 29°C for 4 h. After 4 h, the mixture was centrifuged at 1700 g for 3 min, and the supernatant was discarded. The streptavidin beads were then washed three times with 5 mL of PBS, followed by three washes with 5 mL of deionized water to remove SDS and non-specifically adsorbed proteins.

[0093] Alkylation step: Transfer streptavidin beads to a 1.5 mL screw-top Eppendorf tube and add 500 μL of 6 M Urea in PBS. Then add 10 mM DTT and react at 37 °C for 30 min. Next, add 20 mM IAA and react at 35 °C for 30 min to ensure that all disulfide bonds opened by DTT react with IAA, preventing the re-reinstatement of disulfide bonds.

[0094] Steps for quantifying the three lipoylation modifications of ODP2 in Escherichia coli

[0095] K12 *E. coli* cells stored at -80°C were removed and lysed by sonication with 1.2% SDS in ABS solution. The cells were centrifuged at 20,000g for 1 hour at 20°C, and the supernatant was collected. Protein concentration was determined using a protein quantification kit and adjusted to 8 mg / mL. Three protein samples were prepared: two groups (BAP group) were reacted with 10 mM TCEP for 30 min, followed by the addition of 20 mM BAP probe. The reaction was carried out at 37°C for 12 hours. The other group (DMSO group) was reacted with 10 mM TCEP for 30 min, followed by the addition of an equal volume of DMSO as a control. Subsequent precipitation, click chemistry reaction, streptavidin bead enrichment, and alkylation steps were then performed.

[0096] Enzyme digestion steps: Add 200 μL of 1M Urea PBS, 2 μg of trypsin enzyme, and 2 μL of 100 mM CaCl2 solution to the system, and digest at 37°C for 16 h. After 16 h, wash three times with 1 mL of 100 mM ammonium bicarbonate, and then wash three times with 1 mL of deionized water.

[0097] The dimethylation steps are as follows: The three protein samples are labeled as light-labeled, medium-labeled, and heavy-labeled; HCHO and NaBH3CN are added to the light-labeled samples; DCDO and NaBH3CN are added to the medium-labeled samples; and D2CO3 is added to the heavy-labeled samples. 13 CDO and NaBD3CN reacted completely. The three groups of samples were washed sequentially with TEAB and deionized water. Streptomycin beads were mixed in a 1:1:1 ratio and then washed.

[0098] Acid cutting steps: Add formic acid-mass spectrometer water for acid cutting, and after complete acid cutting, combine all samples.

[0099] The steps for secondary enzyme digestion are as follows: After the sample is dried by rotary evaporation, add 20 μL of 100 mM ammonium bicarbonate and 4 μg of endopeptide V8 protease. After digestion at 37°C for 12 h, perform acid digestion. After acid digestion, dry the sample by rotary evaporation and perform mass spectrometry analysis.

[0100] Example 5: Discovery of a partial compensation mechanism among the three thiocylation modification sites in ODP2

[0101] The number of lipoylation domains in ODP2 varies from one to three across different species (e.g., three in *E. coli* and two in humans). Previous studies have shown that reducing the number of lipoyl domains through genetic engineering slows the growth of *E. coli* in basal media containing different carbon sources (e.g., glucose, lactic acid, succinic acid, acetic acid). On the other hand, studies have shown that one lipoyl domain is sufficient for ODP2 to exercise catalytic activity in vitro. Another interesting question is whether the three lipoylation sites in ODP2 are interdependent or independently regulated.

[0102] To explore the aforementioned scientific questions, the applicant knocked out endogenous ODP2 (ΔODP2) from strain K12 and exogenously expressed wild-type (“WT-ODP2”) and mutant ODP2. The mutant ODP2 included single mutations of three lipoyl lysine derivatives, K41, K144, and K245, each mutated to R, and double-mutant combinations of these derivatives (named “K41R-ODP2”, “K144R-ODP2”, “K245R-ODP2”, “K41R / K144R-ODP2”, “K41R / K245R-ODP2”, and “K144R / K245R-ODP2” strains, respectively). The amino acid numbering followed the Kabat numbering system. “Kabat numbering” refers to the numbering system described by Kabat et al., which is documented in the U.S. Department of Health and Human Services, “Sequence of Proteins of Immunological Interest” (1983).

[0103] Then, the levels of each thiocylation modification site in ODP2 were compared between wild-type and mutant strains using established quantitative methods for thiocylation modification (see [link]). Figure 5 (b) For example Figure 5 As shown in Figure c, the results indicate that in the single mutant, the lipoylation modification at the mutated site disappeared without affecting the lipoylation modification levels at the other two sites. In contrast, in the double mutant, in addition to the disappearance of lipoylation modification at the two mutated sites, the remaining unmutated sites unexpectedly showed increased lipoylation modification. These results suggest that there is a partial compensation mechanism among the three lipoylation modification sites in ODP2.

[0104] Steps for quantifying the three lipoylation modifications of ODP2 in Escherichia coli:

[0105] All steps are largely the same as those in Example 4, specifically the step of "quantifying the three thioctylation modifications of ODP2 in E. coli". The only difference is the dimethylation quantification step. The lightly labeled samples are designated as "K41R-ODP2", "K144R-ODP2", "K245R-ODP2", "K41R / K144R-ODP2", "K41R / K245R-ODP2", and "K144R / K245R-ODP2", while the heavily labeled sample is designated as "WT-ODP2". The two groups of protein samples are labeled as lightly and heavily labeled. HCHO and NaBH3CN are added to the lightly labeled sample; HCHO and NaBD3CN are added to the heavily labeled sample. After the reaction is complete, both groups of samples are washed sequentially with TEAB and deionized water. Streptomycin beads are mixed at a 1:1 ratio and then washed again. The remaining steps are completely consistent with Example 4.

[0106] Example 6: Exploring the function of thioctoyl synthase in Escherichia coli

[0107] The biosynthetic pathway of lipoylation involves three independent lipoyl synthases: octanoyltransferase (LipB) and lipoyl synthase (LipA) in the de novo pathway, and lipoate-protein ligase A (LplA) in the direct pathway. LipB catalyzes the transfer of the octanoyl group from octanoyl-ACP to the substrate protein, followed by LipA inserting a sulfur atom into the octanoic acid in the de novo pathway, resulting in lipoylation. Conversely, LplA functions alone in the direct pathway, catalyzing the transfer of lipoic acid to the substrate protein via the lipoyl-AMP intermediate (see [link to relevant documentation]). Figure 6 (a) To assess the effect of each thiocyl synthase on thiocylation modification, the applicant constructed corresponding knockout strains (ΔLipB, ΔlipA, and ΔlplA) based on WT-K12 and performed a quantitative method for thiocylation modification, comparing the level of thiocylation modification at each known substrate site between the knockout and wild-type strains.

[0108] like Figure 6 As shown in Figure b, knocking out LplA does not significantly affect lipoylation modification. Conversely, knocking out lipoylation modification at all substrate sites in LipA disappears. These results suggest that although the two pathways operate in parallel in bacteria, the de novo synthetic pathway appears to be more important than the direct synthetic pathway in the biosynthesis of lipoylation modification.

[0109] Although LipB and LipA are known to function in tandem in de novo synthetic pathways, knockout of LipB only partially affects lipoylation modifications on all substrates. These data have led to speculation that an alternative pathway may exist in bacteria that bypasses LipB's function and directly produces lipoylated proteins. Literature reports that LplA in the lipoylation pathway can catalyze the transfer of octanoic acid to substrate proteins in vitro using ATP as a substrate, and that octanoic acid supplementation in culture medium may lead to lipoylation of the lipoic acid domain in strains with mutations in both LipA and LipB.

[0110] To further test the contribution of LplA as an octyltransferase in the de novo synthesis pathway, the applicant further constructed a double knockout strain of LipB and LplA (ΔLipB-LplA) to block all possible pathways for the production of octyltransferase proteins. Figure 6As shown in Figure b, quantitative results indicate that thiocylation modifications at all substrate sites were eliminated in the double knockout strain, which supports the view that LplA can replace LipB as an octyltransferase in the de novo synthetic pathway.

[0111] Given that caprylic acid can be utilized by LipB and LplA in de novo synthetic pathways, the applicant next assessed whether caprylic acid might affect these two caprylate transferases. Wild-type and mutant strains were cultured in a medium with glucose as the sole carbon source, and metabolomics analysis revealed that caprylic acid levels in *E. coli* were significantly reduced under these nutrient conditions. Figure 6 As shown in Figure c, the quantitative analysis of lipoylation modification using chemical proteomics, consistent with previous results, revealed that in glucose-only carbon sources, knockout of LipA completely eliminated substrate lipoylation modification, while knockout of LplA minimally disturbed it. However, unlike under perfect nutrient conditions, LipB knockout resulted in the complete loss of lipoylation modification at all sites. Furthermore, exogenous octanoic acid supplementation restored lipoylation modification in the ΔLipB strain. These data collectively indicate that the de novo synthetic pathway plays a dominant role in maintaining normal lipoylation modification levels in bacteria. When LipB is absent, LplA can act as an octanoyltransferase, provided that octanoic acid is adequately supplied in the environment.

[0112] Steps for quantifying the three lipoylation modifications of ODO2 and GCSH in Escherichia coli:

[0113] All steps are largely the same as those in Example 4, specifically the step of "quantifying the thiocylation modification of ODO2 and GCSH in E. coli". The only difference is the dimethylation quantification step. The lightly labeled samples are designated as "ΔLipB", "ΔlipA", "ΔlplA", and "ΔLipB-LplA", while the heavily labeled sample is designated as "WT-K12". The two groups of protein samples are labeled as lightly and heavily labeled. HCHO and NaBH3CN are added to the lightly labeled samples; HCHO and NaBD3CN are added to the heavily labeled samples. After the reaction is complete, both groups of samples are washed sequentially with TEAB and deionized water. Streptomycin beads are mixed at a 1:1 ratio and then washed again. The remaining steps are completely consistent with those in Example 4.

[0114] Steps for quantifying the three lipoylation modifications of ODP2 in Escherichia coli:

[0115] All steps are largely the same as those in Example 4, specifically the step of "quantifying the three thioctylation modifications of ODP2 in E. coli". The only difference is the dimethylation quantification step. The lightly labeled samples are designated as "ΔLipB", "ΔlipA", "ΔlplA", and "ΔLipB-LplA", while the heavily labeled sample is designated as "WT-K12". The two groups of protein samples are labeled as lightly and heavily labeled. HCHO and NaBH3CN are added to the lightly labeled samples; HCHO and NaBD3CN are added to the heavily labeled samples. After the reaction is complete, both groups of samples are washed sequentially with TEAB and deionized water. Streptomycin beads are mixed at a 1:1 ratio and then washed again. The remaining steps are completely consistent with those in Example 4.

[0116] Example 7: Site-specific quantification of lipoylation modifications in the human proteome

[0117] Six known lipoylation modification sites exist in five proteins in the human proteome. Four of these have corresponding sites in *E. coli*: two in the dihydrolipoyllysine acetyltransferase (mammalian cells) (DLAT) protein (a homolog of ODP2), one in the dihydrolipoyllysine succinyltransferase (mammalian cells) (DLST) protein (a homolog of ODO2), and one in the GCSH protein. Unlike *E. coli*, the human proteome has two additional lipoylation modification sites: one in the pyruvate dehydrogenase protein X (PDHX) fraction and the other in the lipoamide acyltransferase (DBT) fraction of the branched α-keto acid dehydrogenase complex.

[0118] When using the same TOP-ABPP method as that used for E. coli to analyze thioctylated modification sites in human cell lysates using standard chemical proteomics procedures, this method can only identify the K107 site in GCSH (see [link to TOP-ABPP method]). Figure 5 (a) The applicant discovered that most peptide sequences containing thiocylation modification sites in the human proteome contain two acidic amino acids, which may affect the detection sensitivity of LC-MS / MS analysis in positive ion mode.

[0119] To investigate whether introducing more positively charged moieties, such as amino groups, could effectively counteract acidic carboxyl groups and improve the ionization efficiency of thiocyl-modified peptides, the applicant designed and synthesized a DADPS-based variant acid-cleavable tag in four steps using biotin-PEG-NHS and Fmoc-lys-N3, named CY58 (see [link to article]). Figure 7 b), after being cleaved by formic acid, has an additional amino group compared to DADPS.

[0120] like Figure 7 As shown in Figure a, when CY58 was introduced into the workflow to replace DADPS, all six lipoylation modification sites in the human proteome were well identified. The applicant then knocked down the level of the lipoyl synthase LIAS in human cells using RNAi technology and quantitatively assessed how its genetic perturbation would affect the level of lipoylation modifications on substrate proteins (see Figure a). Figure 7 (c) First, the applicant confirmed the decrease in mRNA levels using real-time reverse transcription polymerase chain reaction (qPCR). Figure 7 As shown in Figure d, quantitative chemical proteomics results indicate that knockdown of LIAS reduced the modification levels of five known lipoylation sites in DLAT, DLST, DBT, and GCSH. Unfortunately, the K95 lipoylation site in PDHX was not quantified in these experiments, possibly due to its low modification stoichiometry.

[0121] Given that BAP can selectively label endogenous thioacrylation modifications in the proteome, it is compatible with resolvable quality tagging strategies for monitoring the chemometry of thioacrylation modifications on substrate proteins. As a proof-of-concept experiment, the applicant applied a BAP probe and an azide-PEG quality tag with a molecular weight of approximately 2,000 Da (azide-2k-PEG), and resolved the protein chemometry on endogenous GCSH via SDS / PAGE. After treating lysates from K562 and HepG2 cells labeled with thioacrylation modifications with BAP, the azide-2k-PEG quality tag was bound to the thioacrylated protein via a copper-catalyzed click chemistry reaction, and the samples were analyzed using an anti-GCSH antibody-based immunoblotting. Results showed a significant band shift in GCSH, which was attributed to the quality tag-labeled thioacrylation modification (see [link to study]). Figure 7 Based on band intensity, the stoichiometry of GCSH lipoylation modification was measured, finding that the stoichiometry of GCSH lipoylation modification in K562 cells was 62%, while that in HepG2 cells was 30% (see [reference]). Figure 7(f). The reasons for the cell type-specific differences in the stoichiometry of thiocylation modification remain to be studied.

[0122] Quantitative steps for lipoylation modification in human cell lines:

[0123] Human cell lines frozen at -80°C were removed and lysed in 1.2% SDS-ABS solution using sonication. After centrifugation at 20000g for 1 h at 20°C, the supernatant was collected, and the protein concentration was determined using a protein quantification kit and adjusted to 10 mg / mL. After reacting with 10 mM TCEP for 30 min, 20 mM BAP was added. The reaction was carried out at 37°C for 12 h. Unlike the previous method used in *E. coli*, the acid digestion enrichment tag used here was CY58. Subsequent precipitation, click chemistry reaction, streptavidin bead enrichment, and alkylation steps were then performed.

[0124] Before enzyme digestion, add 200 μL of 1M Urea in PBS to the system, then add 2 μg of Trypsin enzyme and digest at 37°C for 16 h. After 16 h, wash three times with 1 mL of PBS, then wash three times with 1 mL of deionized water. Add 100 μL of 100 mM ammonium bicarbonate solution containing 1 M Urea to the system, then add 2 μg of V8 protease and digest at 37°C for 12 h. After 12 h, wash three times with 1 mL of 100 mM ammonium bicarbonate, then wash three times with 1 mL of deionized water.

[0125] The quantitative analysis of dimethylation was performed as follows: Lightly labeled samples were designated "siLIAS", and heavily labeled samples were designated "si-negativecontrol". The two groups of protein samples were labeled as lightly and heavily labeled. HCHO and NaBH3CN were added to the lightly labeled samples; HCHO and NaBD3CN were added to the heavily labeled samples. After the reaction was complete, both groups of samples were washed sequentially with TEAB and deionized water. Streptomycin beads were mixed at a 1:1 ratio and washed again. 200 μL of 2% formic acid-MS / water solution was added for acid digestion for 1 h, repeated twice. The supernatants from the acid digestion were combined, evaporated to dryness, and stored at -20℃ for mass spectrometry analysis.

[0126] Example 8: Synthesis steps of CY58

[0127] The synthesis steps of CY58:

[0128]

[0129] The synthetic route for the CY58 tag and the NMR (nuclear magnetic resonance) and MS (mass spectrometry) confirmation of the final product are described in "Compound Synthesis and Characterization". Reagents and conditions: (a) Et3N, DCM, 0°C to room temperature, 12 h; (b) DCC, HOBT, DCM, rt, 5 h; (c) Ph2SiCl2, pyridine, room temperature, 12 h, followed by CY56, room temperature, 24 h; (d) piperidine, 0°C to room temperature, 5 h.

[0130] ①Chemical synthesis of CY53

[0131] CY52 (77 mg, 0.87 mmol) was added to a solution of compound CY51 (500 mg, 0.85 mmol) in CH2Cl2 (5 mL), followed by Et3N (121 μL, 0.87 mmol). The solvent was evaporated under reduced pressure, and the residue was absorbed into CH2Cl2 and purified by silica gel chromatography with CH2Cl2:MeOH ratios of 15:1 to 5:1 to give CY53 (330 mg, 69% yield).

[0132] 1 H NMR(400MHz,Chloroform-d)δ7.18(s,1H),6.99(s,1H),6.33(s,1H),5.39(s,1H),4.52(dd,J=7.5,4.8Hz,1H),4 .33(dd,J=7.4,4.8Hz,1H),3.76(t,J=5.8Hz,2H),3.65(d,J=3.2Hz,12H),3.57(t,J=4.9Hz,2H),3.44(d,J=4.5H z,2H),3.27(d,J=5.9Hz,2H),3.15(q,J=7.2Hz,1H),2.91(dd,J=12.8,4.8Hz,1H),2.73(d,J=18.4Hz,1H),2.52( t,J=5.8Hz,2H),2.24(t,J=7.4Hz,3H),1.70(dt,J=13.8,6.5Hz,4H),1.43(dt,J=14.5,7.4Hz,2H),1.21(s,6H). 13 C NMR(101MHz,Chloroform-d)δ173.56,172.32,155.17,70.70,70.40,70.22,70.02,69.97,67.51,61.83,60 .19,55.58,50.80,50.31,40.54,39.18,37.05,35.79,28.16,28.06,27.11,27.08,25.62.HRMS(ESI):Calcd for C25 H 47 N4O8S[M+H] + 563.31091, found 563.31071.

[0133] ②Chemical synthesis of CY56

[0134] Add CY55 (500 mg, 1.27 mmol), 1-hydroxybenzotriazole hydrate (HOBt) (193 mg, 1.27 mmol), and CH2Cl2 (25 mL) to a flask. Cool the white suspension to 0 °C. Then add DCC as a solid to the suspension. Stir the reaction mixture at 0 °C for 30 minutes. Then add CY54 (140 μL, 1.52 mmol). Warm the reaction mixture to room temperature and stir overnight. Filter the reaction mixture through a funnel. Wash the organic layer with a saturated solution. Add NaHCO3 (15 mL) and brine (20 mL × 2). Dry the organic layer with Na2SO4, filter, and concentrate under vacuum. Purify by column chromatography (CH2Cl2:MeOH = 20:1 then 10:1) to provide a white solid product (360 mg, yield 61.3%). See also Figure 8 , 1 H NMR(400MHz,Chloroform-d)δ7.76(d,J=7.4Hz,2H),7.57(d,J=7.2Hz,2H),7.35(dt,J=37.7,7.1Hz,4H),6.72(s,1H),5.64(d,J= 7.7Hz,1H),4.50–4.31(m,2H),4.24–4.03(m,2H),3.61(s,2H),3.26(s,4H),2.40(s,1H),2.00(s,3H),1.82(s,1H),1.57(s,7H). 13 C NMR(101MHz,Chloroform-d)δ171.65,156.38,143.70,141.30,127.79,127.11,124.98,120.03,6 7.06,62.19,54.85,51.14,47.10,39.34,32.19,29.64,28.47,26.02,22.72.HRMS(ESI):Calcdfor C 25 H 32 N5O4[M+H] + 466.24488, found466.24483.

[0135] ③Chemical synthesis of CY57

[0136] Ph₂SiCl₂ (1.42 mL, 3.95 mmol, 5 eq) and Et₃N (0.83 mL, 10.37 mmol, 13 eq) were added to 15 mL of CH₂Cl₂ in an ice bath. Compound CY₅₃ (443 mg, 0.79 mmol, 1 eq) was added dropwise to 1.5 mL of CH₂Cl₂. The reaction mixture was then heated to room temperature. After 12 hours, CY₅₆ (4.46 g, 9.32 mmol, 12.2 equivalences) was added and the mixture was stirred for 24 hours. The solution was extracted with CH₂Cl₂, and the organic layer was washed with brine (20 mL × 3). The solvent was dried over Na₂SO₄ and concentrated under vacuum below 30 °C. Purification by column chromatography (MeOH:CH₂Cl₂ = 0:1 to 1:5) gave product CY₅₇ as a white solid (320 mg, 34%). See also Figure 9 , 1 H NMR(400MHz,Chloroform-d)δ7.75(d,J=7.5Hz,2H),7.60(t,J=6.5Hz,5H),7.44–7.26(m,13H),6.95(s,1H), 6.80(s,1H),6.65(t,J=5.7Hz,1H),6.30(s,1H),5.96(d,J=8.4Hz,1H),5.54(s,1H),4.45–4.33(m,2H),4.20( m,2H),3.77–3.49(m,22H),3.42–3.20(m,8H),3.12–3.06(m,1H),2.83(dd,J=12.8,4.9Hz,1H),2.67(d,J=12 .7Hz,1H),2.44(t,J=5.9Hz,3H),2.17(t,J=7.4Hz,2H),1.39(dd,J=12.8,5.6Hz,5H),1.24(d,J=8.5Hz,11H). 13C NMR(151MHz,Chloroform-d)δ173.73,172.43,171.69,171.21,164.06,156.34,143.73,141.29, 135.82,134.41,129.95,127.74,127.11,125.05,120.01,70.76,70.34,70.16,69.97,69.88,67 .50,67.03,62.16,61.87,60.21,55.62,54.83,51.15,50.27,47.11,40.46,39.32,39.15,36.96 ,35.69,32.24,29.70,29.66,28.47,28.19,28.04,27.05,25.97,25.52,22.70.HRMS(ESI):Calcd for C 62 H 86 N9O 12 SSi[M+H] + 1208.58804, found 1208.58837.

[0137] ④Chemical synthesis of CY58

[0138] CY57 (320 mg, 0.26 mmol) was added to 6 mL of CH2Cl2 under ice bath conditions, followed by dropwise addition of 20% piperidine (267 μL, 2.93 mmol). The reaction was then heated to room temperature. After the reaction was completed by TLC, the solution was extracted with CH2Cl2, and the organic layer was washed with brine (20 mL × 3). The solvent was concentrated under vacuum, and the product was purified by rapid chromatography (CH2Cl2:MeOH = 1:0 to 5:1) to obtain product CY58 as a pale yellow solid (260 mg). See also Figure 10 , 1H NMR(600MHz,Chloroform-d)δ7.55(d,J=7.3Hz,4H),7.51(s,1H),7.35(t,J=7.2Hz,2H),7.30(t,J=7.3Hz,4H),7.20(s,1H),6.96 (s,1H),6.57(s,1H),6.25(s,1H),5.39(s,1H),4.42–4.35(m,1H),4.25–4.17(m,1H),3.67(dd,J=14.0,7.7Hz,5H),3.59–3.45(m, 17H),3.41(s,2H),3.37–3.32(m,2H),3.25(d,J=5.9Hz,2H),3.22–3.15(m,5H),3.05(q,J=6.9Hz,1H),2.80(dd,J=12.7,4.7Hz,2 H),2.64(d,J=12.8Hz,2H),2.38(t,J=6.0Hz,3H),2.15(t,J=7.3Hz,2H),1.77(dp,J=15.5,5.5Hz,3H),1.61(s,3H),1.18(s,15H). 13 C NMR(151MHz,Chloroform-d)δ172.76,172.48,170.33,162.75,133.82,133.24,12 9.23,126.87,74.72,69.37,69.35,69.31,69.19,68.94,66.37,61.82,60.81,59. 12,54.57,53.72,50.17,49.44,39.51,38.09,37.85,36.01,34.70,32.98,28.68, 27.63,27.07,27.01,26.62,24.99,24.53,21.84,21.68,13.11.HRMS(ESI):Calcd for C 47 H 76 N8O 10 SSi[M+H] + 986.51996, found986.51929.

[0139] Example 9: Mass spectrometry analysis and data processing of lipoylation modification in Escherichia coli and human cells

[0140] In all embodiments of this application, Thermo Fisher's Q-Exactive Plus mass spectrometer was used to collect mass spectrometry samples. The analytical column had an inner diameter of 75 μm and was packed with 1.9 μm C18 (terminated octadecyl silica gel) packing material; the length of the analytical column was 15 cm. The loading column had an inner diameter of 100 μm and was packed with 5 μm C18 packing material; the length of the loading column was 3 cm. The liquid chromatography flow rate during loading was 3 μL / min. The elution flow rate was 0.3 μL / min. A 166-minute liquid chromatography gradient was used. Specific chromatographic gradient settings are shown in Table 1, and mass spectrometry parameters are shown in Table 2.

[0141] Table 1-Chromatographic gradient at 166 min

[0142]

[0143] Table 2 - Mass Spectrometry Parameter Settings

[0144]

[0145]

[0146] General Setting, Full MS, dd-MS / dd-SIM, Retention Time, Run Time, Polarity, Default Charge, Resolution, Automatic Gain Control (AGC), Maximum Ion Implantation Time (IT), Scan Range, Loop Count, Isolation Window, (N)CE / Stepped Count, Fixed First Mass, Minimum AGC, Dynamic Exclus.

[0147] For mass spectrometry analysis, the mobile phases used were: Phase A was 0.1% formic acid in water, and Phase B was 80% acetonitrile / water (0.1% formic acid). LC-MS / MS data analysis was performed using ProLuCID, with a fixed modification on cysteine ​​residues (+57.0215 Da) and a variable modification on methionine residues (+15.9949 Da). In identifying BSA and E. coli, the variable modification of lipoylation was an increase of 411.20132 Da on lysine. In identifying mammalian cells, the variable modification of lipoylation was an increase of 511.26508 Da on lysine.

[0148] Similar parts between the embodiments provided in this application can be referred to mutually. The specific implementation methods provided above are only a few examples under the overall concept of this application and do not constitute a limitation on the scope of protection of this application. For those skilled in the art, any other implementation methods extended from the solution of this application without creative effort shall fall within the scope of protection of this application.

Claims

1. A quantitative chemical proteomic method for lipoic-modification sites, characterized in that, include: Probes containing aldehyde and alkyne groups were used to chemically selectively label thiocylation modifications in the proteome of biological samples. Quantitative chemical proteomics techniques were used to quantitatively analyze the thioctylation modification sites after chemical selective labeling. The structure of the probe containing aldehyde and alkyne groups is as follows: n = 1-3, where n is an integer.

2. The quantitative chemical proteomics method for thiocylation modification sites according to claim 1, characterized in that, The biological samples include: Escherichia coli, yeast cells, human cell lines, human tissue samples, human organs, mouse cell lines, mouse tissue samples, mouse organs, Drosophila cell lines, zebrafish cell lines, or nematode cell lines.

3. The quantitative chemical proteomics method for thiocylation modification sites according to claim 2, characterized in that, The quantitative analysis includes biological sample pretreatment and mass spectrometry analysis. The biological sample pretreatment steps include: click chemistry reaction of chemically selectively labeled proteins, protein precipitation, streptavidin bead enrichment and alkylation, enzyme digestion, dimethylation quantification, and acid digestion.

4. The quantitative chemical proteomics method for thiocylation modification sites according to claim 3, characterized in that, When the biological sample is *E. coli*, the pretreatment enzyme digestion steps for quantitative analysis of lipo-octylation modification sites in *E. coli* ODO2 and GCSH proteins include: Enzymatic digestion was performed using V8 protease dissolved in ammonium bicarbonate solution.

5. The quantitative chemical proteomics method for thiocylation modification sites according to claim 3, characterized in that, When the biological sample is *E. coli*, the pretreatment enzyme digestion steps for quantitative analysis of the three lipoylation modification sites of the ODP2 protein in *E. coli* include: Trypsin dissolved in phosphate buffer solution was added for enzymatic digestion. After the acid digestion step, V8 protease dissolved in ammonium bicarbonate solution was used for enzymatic digestion again.

6. The quantitative chemical proteomics method for thiocylation modification sites according to claim 3, characterized in that, When the biological sample is a human cell line, the pretreatment enzyme digestion steps for quantitative analysis of all lipoylation modification sites in the human cell line include: Trypsin and V8 protease dissolved in phosphate buffer solution were added simultaneously for enzymatic digestion.

7. The quantitative chemical proteomics method for thiocylation modification sites according to claim 3, characterized in that, When the biological sample is a human cell line, the CY58 acid-cutterable tag is used for cutting during the acid cutting step. The structural formula of the CY58 tag is as follows: 。 8. The quantitative chemical proteomics method for thiocylation modification sites according to claim 7, characterized in that, The synthesis steps of CY58 are as follows: 。 9. The quantitative chemical proteomics method for thiocylation modification sites according to claim 2, characterized in that, When the biological sample is *E. coli*, the quantitative chemical proteomics method for the thioctylation modification sites can identify and quantify five thioctylation modification sites in the *E. coli* proteome; or, When the biological sample is a human proteome sample, the quantitative chemical proteomics method for the thioctylation modification sites can identify and quantify six thioctylation modification sites in the human cell line proteome.

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