High-fidelity biotinylation probe, preparation method thereof and application of high-fidelity biotinylation probe in polysulfide detection

Abstract

The invention belongs to the technical field of biological detection and analysis, and particularly relates to a high-fidelity biotinylation probe as well as a preparation method and application thereof in polysulfide detection. Specifically, the invention develops a novel biotinylation probe HPB, and the probe is formed by coupling HPE-IAM and biotin. Experimental results show that HPB is superior to IAB and other traditional reagents in the aspects of maintaining and efficiently labeling polysulfide, and a high-specificity solution is provided for Pr-SnH analysis at the proteomics level. By optimizing a marking scheme and introducing a precipitation-redissolution step, the detection accuracy of the polysulfide modified protein is further improved, so that a reliable tool is provided for protein polysulfide modification detection research under a complex cell background, and the method has a good practical application value.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological detection and analysis, and particularly relates to a high-fidelity biotinylated probe and a preparation method thereof, and application thereof in polysulfide detection. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance understanding of the overall background of the invention and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to those skilled in the art.

[0003] Polysulfides are essential metabolites with important physiological and pathological functions in prokaryotes and eukaryotes. Common ones include GS n H and Cysteine-S n H (n≥2). As antioxidants, polysulfides can directly scavenge lipid free radicals and reduce the risk of ferroptosis. Polysulfides can also react with protein cysteine thiol (-SH) to form polysulfide-modified proteins (Pr-S n H, n ≥ 2). This post-translational modification (called protein persulfation or polysulfide modification) not only prevents irreversible oxidation of protein thiols but also regulates protein function, thereby affecting various biological processes such as metabolism, transcriptional networks, and ion channel function.

[0004] A variety of polysulfide detection methods have been developed. However, due to the presence of sulfhydryl groups (-S n H), and its chemical similarity to thiols (-SH), subsulfonic acids (-SOH), S-nitrosothiols (-SNO) and disulfide bonds (-SS-), poses a major challenge in distinguishing other modifications of polysulfides. Initially, Mustafa et al. used S-methyl methanethiosulfonate (MMTS) and N-(6-(biotinamino)hexyl)-3'-(2'-pyridyldithio)propionamide (Biotin-HPDP) to detect polysulfide modifications of proteins. This method assumes that MMTS only blocks -SH, while Biotin-HPDP can label -SSH. However, subsequent studies found that MMTS also blocks -S n Subsequently, Zhang et al. and Zivanovic et al. proposed two label conversion methods, using electrophilic reagents such as methylsulfonylbenzothiazole (MSBT) or 4-chloro-7-nitrobenzofuran (NBF-Cl) to label -SH and -S n H group, and then selectively convert the labeled -SSH by CN-Biotin (biotinylated cyanoacetic acid label) or DCP-Bio1 (biotinylated dimedone probe). Although these methods have improved the efficiency, the electrophilic reagents are still not very effective for -SSH. nThe specificity and label conversion efficiency of H still need to be further verified and improved. Gao et al. and Doka et al. proposed the BTA and ProPerDP methods, respectively, which use N-ethylmaleimide (NEM) or iodoacetamide (IAM)-derived biotin tags (NEB and IAB) as alkylating agents to label Pr-S n H, then the biotinylated protein (Pr-SS-biotin) was captured by streptavidin magnetic beads, and finally the Pr-S was cut with dithiothreitol (DTT) or tris (2-carboxyethyl) phosphine (TCEP). n - disulfide bond of biotin to release the -S n The main controversy between these two methods focuses on the selectivity or efficiency of NEM and IAM. Summary of the Invention

[0005] To address the shortcomings of the prior art, the present invention provides a high-fidelity biotinylated probe, a preparation method thereof, and its application in polysulfide detection. Specifically, the present invention develops a novel biotinylated probe, HPB, which is synthesized by coupling HPE-IAM with biotin. Experimental results show that HPB outperforms traditional reagents such as IAB in retaining and efficiently labeling small molecule polysulfides and polysulfide-modified proteins, providing a promising target for proteomics-level Pr-S n H analysis provides a highly specific solution. By optimizing the labeling scheme and introducing a precipitation-redissolution step, the present invention further improves the accuracy of detecting polysulfide-modified proteins, providing a reliable tool for detecting protein polysulfide modifications in complex cellular contexts. Based on these research findings, the present invention was completed.

[0006] Specifically, the present invention relates to the following technical solutions:

[0007] The first aspect of the present invention provides a high-fidelity biotinylated probe having the following structure:

[0008]

[0009] In the present invention, the high-fidelity biotinylated probe is named HPB.

[0010] Specifically, the present invention synthesizes the above-mentioned probe HPB based on the coupling of HPE-IAM and biotin, which has no redox activity to polysulfides and has excellent alkylation specificity, thereby being able to maintain and efficiently label small molecule polysulfides and polysulfide-modified proteins.

[0011] The second aspect of the present invention provides a method for preparing the above-mentioned high-fidelity biotinylated probe, which comprises the following synthetic route:

[0012]

[0013] Furthermore, the preparation method includes:

[0014] S1: Biotin, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI), 4-dimethylaminopyridine (DMAP) and N-tert-butyloxycarbonyl-L-tyrosine were mixed to generate intermediate I, which was purified by silica gel flash column chromatography;

[0015] S2: intermediate I is treated with hydrochloric acid (HCl) to remove the Boc protecting group to obtain intermediate II;

[0016] S3: Intermediate II reacts with diisopropylethylamine (DIPEA) and chloroacetyl chloride to generate intermediate III containing a chlorine atom;

[0017] S4: Intermediate III is reacted with potassium iodide (KI) to replace the chlorine atom with the iodine atom.

[0018] Furthermore, in step S1, the molar ratio of biotin, EDCI, DMAP and N-tert-butyloxycarbonyl-L-tyrosine is 1-5:1-5:10-30:1-5; preferably 2:3:20:2.4;

[0019] In step S2, the molar ratio of the intermediate I to hydrochloric acid is 0.5-5:1-5; preferably 1:2;

[0020] In step S3, the molar ratio of the intermediate II, DIPEA and chloroacetyl chloride is 0.5-5:1-5:1-5; preferably 1.0:2.0:1.2;

[0021] In step S4, the molar ratio of the intermediate III to KI is 1:0.5-5, preferably 1:2;

[0022] By controlling the above dosage ratio, the yield and production rate of the above product can be improved.

[0023] The third aspect of the present invention provides a detection kit, which comprises at least the above-mentioned probe. Further, the detection kit may also comprise any other known reagents (such as reaction enhancers, enzyme reagents, buffers, cleaning solutions), etc., which are not specifically limited here.

[0024] The fourth aspect of the present invention provides the use of the above-mentioned probe or detection kit in the detection of small molecule polysulfides or protein polysulfide modifications.

[0025] Furthermore, the application can be to treat small molecule polysulfides (such as GS) in cells. n H and Cysteine-S nH (n ≥ 2)) and proteome-wide polysulfide-modified protein labeling and screening, i.e., such applications can be performed intracellularly. In one embodiment of the present invention, the cells are HT29 cells. Compared to the commonly used alkylating agent IAB, the probe HPB of the present invention offers significant advantages in reaction specificity and polysulfide chain retention, thus facilitating its practical application, particularly in the detection of protein polysulfide modifications in complex cellular contexts.

[0026] Therefore, a fifth aspect of the present invention provides a method for detecting and analyzing polysulfide-modified proteins in a cell proteome, the method comprising: adding cells to a cell lysis solution containing the above-mentioned probe or detection kit for lysis, thereby achieving in situ labeling;

[0027] However, since high concentrations of small molecule thiols (such as GSH) and polysulfides (such as GSSH) in cells will consume or reduce the alkylation reagent, resulting in low labeling efficiency, preferably, the method is: after adding the cells to the lysis buffer, protein precipitation is performed to remove small molecule interfering substances, and after re-dissolving the protein, the above-mentioned probe or detection kit is added to perform alkylation labeling. This step can effectively improve the enrichment efficiency of polysulfide-modified proteins.

[0028] Furthermore, due to the presence of nonspecific alkylation, the protein obtained by the above method may still be contaminated with non-target modification products. Therefore, it is further preferred to further enrich the peptides after enzymatic digestion of the alkylated protein using streptavidin magnetic beads. This allows the resulting peptides to be classified into two categories: those containing cysteine (specific modification) and those containing non-cysteine (nonspecific modification). This method also demonstrates that the probe HPB used in the present invention has a lower false positive rate.

[0029] Beneficial technical effects of one or more of the above technical solutions:

[0030] The above-mentioned technical scheme successfully synthesized the polysulfide modification detection probe HPB through a four-step reaction. The probe is based on the HPE-IAM structure and is conjugated to biotin. When reacting with glutathione persulfide (GSSH), HPB only exhibited alkylation activity, and no reduced HPB was detected. In contrast, IAB displayed significant redox activity, resulting in the production of reduced IAB. In peptide alkylation experiments, HPB had significantly fewer nonspecific modification sites than IAB. Ultimately, in the labeling and screening of polysulfide-modified proteins in the HT29 cell proteome, HPB produced a lower proportion of false-positive peptides than IAB. These results demonstrate that HPB offers significant advantages over the commonly used alkylating agent IAB in terms of reaction specificity and polysulfide chain retention, making it more suitable for practical applications, particularly in the detection of small molecule polysulfides and protein polysulfide modifications in complex cellular contexts. Therefore, it has excellent practical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] The accompanying drawings, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0032] Figure 1 The synthesis route and structure confirmation of the probe HPB of the present invention; wherein A is the four-step reaction synthesis route; B is the primary mass spectrometry (LC-MS) analysis of HPB; C is the secondary mass spectrometry (LC-MS / MS) analysis of HPB; D is the nuclear magnetic resonance hydrogen spectrum ( 1 1H NMR).

[0033] Figure 2 This is the identification of the reaction products of HPB or IAB with GSSH in the present invention. A represents the reaction product of HPB (3 mM) and GSSH (0.5 mM); the main product is GSS-PB, and the byproducts are GS-PB and GSSS-PB. B represents the reaction product of IAB (3 mM) and GSSH (0.5 mM); the main product is GSS-AB, and the byproducts are GS-AB and GSSS-AB. C and E represent the identification of potential reduction products from the reaction of HPB with GSSH. D and F represent the identification of IAB and GS4G, the byproducts of the reaction of IAB with GSSH.

[0034] Figure 3 Comparison of the reactivity specificity of HPB or IAB with peptide P1 in the present invention. A is the mass spectrum of P1 (100 μM) labeled with HPB (3 mM). B is the mass spectrum of P1 (100 μM) labeled with IAB (3 mM).

[0035] Figure 4 In the present invention, HPB or IAB retains Trx1 m Comparison of the ability of polysulfide modification. A is HPB-labeled Trx1 m Mass spectrum of Trx1. B is IAB-labeled Trx1 m C is the mass spectrum of Trx1 after labeling with different reagents m The relative proportion of non-specificity in the n (1 mM) incubation and HPB (5 mM) labeled Trx1 m (100μM) mass spectrum. E is H2S n (1 mM) incubation and IAB (5 mM) labeled Trx1 m (100μM) mass spectrum. F is H2S n Trx1 after treatment with different reagents m The relative proportion of polysulfide modification in the medium.

[0036] Figure 5 The present invention describes the application of HPB in the analysis of polysulfide modifications in proteomes. A depicts the separation of polysulfide-modified proteins from a proteome using workflows 1 and 2. The difference is that workflow 2 includes a precipitation-dissolution step to remove small molecule thiols and polysulfides. Workflow 3 is used to separate polysulfide-modified peptides from a trypsin-digested proteome. B depicts H2S n Trx1 alkylated by HPB after incubation m Mass spectrum of C is H2S n After incubation, Trx1 was alkylated with HPB after acetone-urea precipitation-solubilization step. m Mass spectra of alkylated Trx1 produced by different treatment steps. m The relative proportions of 1 and 2 are calculated from Figure B and C, respectively. E shows the percentage of persulfated proteins obtained using Workflow 2 when IAB and HPB were used to label proteins, respectively. F shows the difference in the percentage of cysteine-containing peptides obtained using Workflow 3 when IAB and HPB were used to label proteins, respectively. DETAILED DESCRIPTION

[0037] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present application belongs.

[0038] It should be noted that the terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the exemplary embodiments according to the present application. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components and / or combinations thereof. In the following specific embodiments, if the experimental methods for specific conditions are not specified, they are generally in accordance with conventional methods and conditions within the art, and such techniques and conditions are fully explained in the literature.

[0039] The present invention will be further described with reference to specific examples. The following examples are intended only to illustrate the present invention and are not intended to limit its contents. Experimental conditions not specified in the examples are generally based on conventional conditions or those recommended by the sales company. Materials and reagents used in the examples are commercially available unless otherwise specified.

[0040] The present invention is further explained by the following examples, but is not intended to limit the present invention. It should be understood that these examples are only intended to illustrate the present invention and are not intended to limit the scope of the present invention.

[0041] Example

[0042] 1. Materials and Methods

[0043] 1.1 Strains, cell lines, and reagents

[0044] The strains, human cell lines, and plasmids used in the experiments are detailed in Table 1. Escherichia coli strains were cultured in LB medium at 37°C with shaking at 200 rpm. HT29 cells (purchased from ATCC) were cultured in McCoy's 5A medium supplemented with 10% fetal bovine serum at 37°C with 5% CO2.

[0045] Biotin, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI), 4-dimethylaminopyridine (DMAP), tert-butyloxycarbonyltyrosine (BOC-L-Tyrosine), dichloromethane (DCM), ethyl acetate (EAC), diisopropylethylamine (DIPEA), chloroacetyl chloride (DMF), potassium iodide (KI), reduced glutathione (GSH), elemental sulfur (S8, purity 99.9%), and sodium hydrosulfide (NaHS) were purchased from Sigma-Aldrich (Shanghai, China). TM Iodoacetyl-PEG2-biotin (IAB) was purchased from Thermo Fisher Scientific (Shanghai, China). Preparation of S8 solution: Excess sulfur powder was dissolved in acetone to prepare a saturated solution, and its concentration was determined to be 17 mM by cold cyanidation method. Hydrogen polysulfide (H2S n ) solution was prepared according to standard method.

[0046] Table 1. Strains and plasmids used in this example

[0047]

[0048]

[0049] 1.2 Synthesis of HPB

[0050] Step 1: Biotin (2.0 mM), EDCI (3.0 mM), and DMAP (20 mM) were sequentially added to a round-bottom flask containing anhydrous DCM (0°C). The mixture was stirred overnight until the biotin reaction was complete. A solution of BOC-L-tyrosine (2.4 mM) in DCM was then slowly added dropwise at room temperature. The reaction progress was monitored by thin-layer chromatography (TLC). After completion of the reaction, the mixture was diluted with DCM and extracted. The organic phases were washed sequentially with saturated sodium bicarbonate and saturated brine, combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography.

[0051] Step 2: Under argon, dissolve the intermediate (1.5 mM) in 5 mL of ethyl acetate, add 3 mL of 3N HCl, and stir at room temperature. Monitor the reaction progress by TLC. Upon completion, dilute with ethyl acetate, wash sequentially with saturated sodium bicarbonate and brine, dry, and concentrate. The crude product is purified by flash column chromatography.

[0052] Step 3: Under argon, dissolve the intermediate (1.0 mM) in DCM and add DIPEA (2.0 mM). After cooling the solution to 0°C, slowly add chloroacetyl chloride (1.2 mM) dropwise, maintaining the temperature between 0 and 10°C. The reaction mixture is diluted with ethyl acetate and extracted, washed sequentially with saturated sodium bicarbonate and brine, dried, and concentrated. The product is purified by flash column chromatography.

[0053] Step 4: Under argon, the intermediate (0.5 mM) was dissolved in 2 mL of DMF, and KI (1.0 mM) was added. The reaction was allowed to react at room temperature until the starting material was completely consumed. The reaction solution was diluted with water and extracted with ethyl acetate. The mixture was washed sequentially with saturated sodium bicarbonate and brine, dried, and concentrated. The crude product was purified by flash column chromatography to obtain the desired product, HPB.

[0054] 1.3 Reaction of IAB / HPB with GSSH

[0055] GSSH was prepared by mixing equal volumes of 17 mM GSH (dissolved in 100 mM potassium phosphate buffer, pH 7.4) and 17 mM saturated sulfur solution (concentration determined by cold cyanidation). To 100 μL of 0.5 mM GSSH, 30 μL of 10 mM IAB (dissolved in potassium phosphate buffer, pH 8.0) or HPB (dissolved in methanol) was added and incubated at 37°C in the dark for 40 minutes. After reaction, the sample was centrifuged at 12,000 × g for 3 minutes, and the supernatant was analyzed by liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS).

[0056] 1.4 LC-ESI-MS analysis

[0057] The analytical method was referred to the literature. Brief procedure: Samples were separated on an InertSustain C18 column (Shimadzu, Japan) and coupled to a high-resolution Q-TOF mass spectrometer (Ultimate 3000, Bruker impact HD, Germany). Mobile phase A consisted of 0.25% acetic acid in water, and mobile phase B consisted of 100% methanol. Gradient elution program: Phase B increased from 7.5% to 52.5% at 0-1 min; maintained at 52.5% at 1-15 min; increased to 55% at 15 min, then increased to 100% and held for 5 min; then decreased to 7.5% at 20.1 min and continued until the end of the analysis at 31 min. The electrospray ionization (ESI) source was operated at 200°C, a spray voltage of 4.5 kV, and nitrogen was used as the nebulizer and drying gas. Data were processed using Data Analysis 4.2 software.

[0058] 1.5 Reaction of IAB / HPB with peptides

[0059] Peptides P1 and P2 were synthesized by GenScript Biotech (Nanjing, China). 1 mg of peptide powder was dissolved in 1 mL of ultrapure water to prepare a 0.3 mM solution, which was further diluted to 0.1 mM with PBS. 200 μL of the peptide solution was added with 0.4 μL of TCEP (final concentration 0.2 mM) and incubated at room temperature for 10 minutes to ensure the peptides were in the reduced state.

[0060] No H2S n Treatment group: The peptide solution was divided into two portions (100 μL each), and 12 μL of 25 mM IAB or HPB (final concentration 3 mM) was added to each portion, and the mixture was reacted at 37° C. in the dark for 40 minutes.

[0061] H2S n Treatment group: 10 μL of 20 mM H2S was added to 200 μL of 0.1 mM peptide solution. n The samples were divided into two equal parts, and 20 μL of 25 mM IAB or HPB (final concentration 5 mM) was added to each part, and the reaction was carried out under the same conditions for 40 minutes.

[0062] After the reaction, salts and small molecule impurities were removed using a C18 spin column (Thermo Fisher Scientific, China). The purified peptides were dissolved in ultrapure water and analyzed by LC-MS.

[0063] 1.6 Protein expression and purification

[0064] Will carry pET30-Trx1 m E. coli BL21 (DE3) expressing the plasmid was inoculated into LB medium containing 100 μg / mL kanamycin and cultured. 600 When the protein concentration reached 0.6–0.8, expression was induced by adding 0.3 mM IPTG and cultured at 20°C for 18 hours. Bacteria were harvested by centrifugation and resuspended in lysis buffer (50 mM NaH2PO4, 300 mM NaCl, 20 mM imidazole, 0.5 mM DTT, pH 8.0). After disruption using an SPCH-18 high-pressure homogenizer, the supernatant was centrifuged at 12,000 × g for 15 minutes. The supernatant was purified using Ni-NTA agarose affinity resin and exchanged with a desalting column (PD-10, GE Healthcare) into desalting buffer (0.5 mM DTT, 50 mM Tris-HCl, 10% glycerol, pH 7.4). The purified protein was stored in an anaerobic incubator on ice until use. Protein purity was verified by SDS-PAGE, and concentration was determined by BCA assay (Beyotime Biotechnology, China).

[0065] 1.7 Reaction of IAB / HPB with Trx1m

[0066] Reduced Trx1 m (100 μM) was incubated with 200 μM DTT at room temperature for 10 minutes, and 0.5 μL 100 mM TCEP was added and incubated for another 10 minutes. Subsequently, 10 μL 20 mM H2S was added. n The reaction was carried out at room temperature for 30 minutes. The sample was divided into two parts, 30 μL of 25 mM IAB or HPB was added to each part, and the reaction was carried out at 37°C in the dark for 40 minutes. The unreacted reagent was washed with 3K Ultrafiltration centrifuge tubes (Sigma-Aldrich, China) were used for removal, and the molecular weight changes were analyzed by LC-MS.

[0067] To investigate the effect of precipitation-redissolution steps on polysulfide modification, H2S n Processed Trx1 m Divided into two groups:

[0068] Direct labeling group: After adding HPB, incubate at 37°C in the dark for 40 minutes. Then, add 3 volumes of pre-chilled acetone and precipitate at -20°C for 1 hour. Centrifuge and discard the supernatant. Redissolve in 100 μL of 8 M urea (pH 7.4-8.0), desalt, and analyze.

[0069] Post-precipitation labeling group: first add 3 volumes of pre-cooled acetone to precipitate the protein, redissolve in 100 μL 8 M urea and 20 μL HPB (25 mM), shake at 37°C (600 rpm) for 40 minutes, and analyze after desalting.

[0070] 1.8 LC-MS analysis of peptides and proteins

[0071] The intact protein molecular weight was analyzed using an LC-MS system equipped with an XBridge Protein BEH C4Sentry Guard column (Waters TM , USA) and a high-resolution Q-TOF mass spectrometer (Ultimate 3000, Bruker impact HD, Germany). Mobile phase A consisted of 0.1% formic acid in water, and mobile phase B consisted of a mixture of acetonitrile and 0.1% formic acid. The gradient program was as follows: 0-7 minutes, with phase B maintained at 5%; at 7 minutes, the gradient was switched to 10% 0.1% formic acid + 90% acetonitrile / 0.1% formic acid, maintained for 3 minutes; at 10 minutes, phase B was returned to 5% for 3 minutes. The flow rate was constant at 0.5 mL / min. Mass spectrometry was performed in positive electrospray ionization (ESI) mode with a capillary voltage of 3500 V. Data were deconvoluted and analyzed using Data Analysis 4.2 software.

[0072] 1.9 Detection of polysulfide-modified proteins in HT29 cells

[0073] Take HT29 cells (10 cm culture dish) grown to 80-90% confluence, discard the culture medium and wash with PBS, trypsinize for about 3 minutes, collect the cell suspension into 2 mL centrifuge tube (about 1×10 7 Cells were harvested by centrifugation at 200 × g for 5 minutes at 4°C, washed three times with PBS, and resuspended in 400 μL of RIPA lysis buffer (containing 1% protease inhibitors and 5 mM IAB or HPB, pH 7.6) and lysed on ice for 5 minutes. 1.6 mL of pre-chilled acetone was added and the cells were precipitated at -20°C for 1 hour. The supernatant was discarded after centrifugation at 13,000 × g for 10 minutes at 4°C. The pellet was washed twice with 100% acetone, dried in acetone, and incubated at 37°C with shaking in the dark for 50 minutes. The reaction solution was desalted using a 10 kDa ultrafiltration tube and exchanged into TBS buffer (20 mM Tris, 0.137 M NaCl, pH 7.6). Protein concentration was determined using the Bradford assay (Beyotime Biotechnology, China).

[0074] 2 mL of streptavidin magnetic beads were aliquoted into two tubes. The supernatant was discarded on a magnetic rack and washed with TBS. 350 μg of protein was incubated with 2 mL of magnetic beads at room temperature for 1 hour (170 μg protein / 1 mL of magnetic beads). The supernatant was discarded on a magnetic rack and washed six times with TBS. 100 μL of 8 mM TCEP (dissolved in 50 mM Tris-HCl, pH 7.0) was added and shaken at room temperature in the dark for 30 minutes. The eluate was collected on a magnetic rack, separated by 12% SDS-PAGE, and visualized using a fast silver staining kit (Beyotime Biotechnology, China).

[0075] 1.10 Detection of polysulfide-modified peptides

[0076] The steps of cell collection, lysis and acetone precipitation were the same as above. After the protein was labeled with IAB or HPB, it was stained with 3K Ultrafiltration tubes (Sigma-Aldrich, China) were desalted and replaced with 25 mM NH₄HCO₃ buffer. After protein concentration was determined by Bradford assay, 350 μg of protein was digested with trypsin (enzyme:protein = 1:25) at 37°C overnight (12-16 hours). The next day, the reaction was terminated by heating at 95°C for 10 minutes and incubated with 2 mL of streptavidin magnetic beads at room temperature with shaking for 1 hour (170 μg peptide / 1 mL beads). The supernatant was discarded from the magnetic rack and washed six times with TBS. 100 μL of 8 mM TCEP (dissolved in 50 mM Tris-HCl, pH 7.0) was added and shaken at room temperature in the dark for 30 minutes. The eluate was collected and reacted with 15 mM iodoacetamide (IAM) at 37°C in the dark for 30 minutes. Salts were removed by passing through a C18 spin column, and the peptides were eluted with 70% acetonitrile. After vacuum drying, the peptides were reconstituted in 10 μL of ultrapure water for LC-MS analysis.

[0077] 1.11 LC-MS / MS analysis

[0078] A Prominence nanoliter HPLC system (Shimadzu, Shanghai, China) was used in conjunction with a custom-made silica gel column (75 μm × 15 cm, packed with 3 μm Reprosil-Pur 120C18-AQ packing). Mobile phases A and B consisted of 0.1% formic acid / 2% acetonitrile and 0.1% formic acid / 98% acetonitrile, respectively, with a flow rate of 300 nl / min and a gradient elution over 100 min (phase B 0–100%). The eluent was electrospray ionized on an LTQ-Orbitrap Velos Pro CID mass spectrometer (Thermo Fisher Scientific, Shanghai, China) using Xcalibur 2.2.0 software in data-dependent acquisition mode. The full-scan mass spectrum range was 400–1800 m / z, with an Orbitrap resolution of 60,000 at 400 m / z. Data were analyzed using Proteome Discoverer 3.2 software.

[0079] 2. Research Results

[0080] 2.1 Synthesis and structure confirmation of HPB

[0081] HPB is synthesized via four-step reaction ( Figure 1 A): In the first step, biotin, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI), 4-dimethylaminopyridine (DMAP) and tert-butyloxycarbonyltyrosine are reacted to produce intermediate I, which is then purified by silica gel flash column chromatography. In the second step, intermediate I is treated with hydrochloric acid (HCl) to remove the Boc protecting group to produce intermediate II. In the third step, intermediate II is reacted with diisopropylethylamine (DIPEA) and chloroacetyl chloride to produce intermediate III containing a chlorine atom. In the fourth step, intermediate III is reacted with potassium iodide (KI) to replace the chlorine atom with an iodine atom. The final product is named HPB.

[0082] The HPB structure was confirmed by mass spectrometry (ESI-Q-TOF) and nuclear magnetic resonance (NMR) analysis. The mass spectrometry detection showed that the m / z value was 532.0745 ( Figure 1 B and 1C), which is consistent with the theoretical molecular weight of HPB; the nuclear magnetic resonance spectrum further confirmed its structural characteristics ( Figure 1 D).

[0083] 2.2 Alkylation of Glutathione Persulfide by HPB and IAB

[0084] Glutathione persulfide (GSSH) was used as a model compound to evaluate the alkylation effect of HPB. GSSH (0.5 mM) was reacted with excess HPB (3 mM) at 37°C for 40 minutes and then analyzed by LC-MS. The results showed that the main product after alkylation was GSS-HPB ( Figure 2 A). Since GSSH is prepared by the reaction of glutathione (GSH) and elemental sulfur (S8) and is inherently unstable, GSH and GSSSH are inevitably present in its aqueous solution. Therefore, a small amount of GS-HPB and GSSS-HPB by-products were detected in the experiment. As a control, when IAB (IAM-derived biotin tag) was used for the same reaction, the main product was GSH-AB, and the minor products were GSS-AB and GSSS-AB ( Figure 2 B).

[0085] Previous studies have shown that halogenated alkylating agents (especially IAM derivatives) can be reduced by small molecular weight polysulfides (reductive dehalogenation), thereby reducing the efficiency of alkylation. This experiment detected the reduction products after the alkylation reaction: HPB and GSSH reaction system did not detect reduced HPB and oxidized GSSH (GS4G) ( Figure 2 C and 2D); while the IAB and GSSH reaction system produces a large amount of reduced IAB and GS4G ( Figure 2 E and 2F). This indicates that HPB has no redox activity towards polysulfides and its alkylation efficiency is significantly better than that of IAB.

[0086] 2.3 Alkylation Specificity of HPB and IAB on Synthetic Peptides

[0087] Previous studies have reported that iodinated alkylating agents can derivatize other amino acid residues in addition to modifying cysteine, leading to nonspecific alkylation. In order to evaluate the alkylation specificity of HPB and IAB, this study synthesized a single cysteine residue (Cys 15 ) of the 21-peptide P1( Figure 3 A). P1 was reacted with HPB and IAB at 37°C in the dark for 40 minutes and then analyzed by LC-MS. The results showed that the main peak of P1 after HPB reaction was 3,782.8Da (corresponding to Cys 15 P1-S-HPB with single HPB alkylation), and a minor peak of 4,185.9 Da (corresponding to two HPB alkylation products) ( Figure 3 A); the main peak of P1 after IAB reaction is 3,793.8Da (corresponding to P1-S-AB with single IAB alkylation at Cys15 site), and the minor peaks of 4,208.1Da, 4,623.2Da and 5,038.4Da correspond to di-, tri- and tetra-IAB alkylation products, respectively ( Figure 3 B) The above results indicate that HPB induces significantly less nonspecific alkylation than IAB.

[0088] 2.4Alkylation of Thioredoxin 1 by HPB and IAB

[0089] Saccharomyces cerevisiae thioredoxin 1 (Trx1) can react with polysulfides to form disulfide bonds (Cys 30 With Cys 33 site) and persulfate modification (Cys 30 In this study, Cys 33 Mutation to serine to retain Cys 30 , ensuring that only persulfation modification occurs. m ) was expressed in E. coli BL21 (DE3) through an N-terminal His tag and purified by Ni column. m After reacting with HPB and IAB, LC-MS analysis was performed. The results showed that HPB-labeled Trx1 m The main peak has a molecular weight of 12,686 Da (+403 Da, corresponding to a single HPB alkylation product), and the secondary peak has a molecular weight of 13,089 Da, corresponding to two HPB alkylation products ( Figure 4 A); IAB-labeled Trx1 m The main peak is 12,697 Da (+414 Da, corresponding to the mono-IAB alkylation product), and the minor peaks 13,112 Da and 13,526 Da correspond to the di- and tri-IAB alkylation products, respectively ( Figure 4 B) Since the N-terminal methionine residue is easily removed during LC-ESI-MS analysis, the experiment also detected the modified form of methionine deletion (Trx1 m -Met-1). Statistics show that HPB monoalkylates protein (Trx1 m -S-PB and Trx1 m -Met-1-S-PB) accounted for 95.0%, while IAB monoalkylated protein (Trx1 m -S-AB and Trx1 m -Met-1-S-AB) accounts for only 79.5% ( Figure 4 C), consistent with the results of the aforementioned peptide experiments, confirming that HPB has better alkylation specificity than IAB.

[0090] Purified Trx1 m Hydrogen polysulfide (H2S n ) were reacted for 30 minutes to form polysulfide modification, and then labeled with HPB or IAB and analyzed by LC-ESI-MS. In the HPB labeled group, the reduced state (-SH direct alkylation) Trx1 m -S-PB and Trx1 m-Met-1-S-PB accounted for the highest proportion; polysulfide modified forms (-SS-PB, -SSS-PB, -SSSS-PB) were also detected, which were generated by HPB alkylation of -SSH, -SSSH, and -SSSSH, respectively, accounting for 66% of the total labeled proteins ( Figure 4 D and 4F). In the IAB labeling group, the reduced state (-SH direct alkylation) of Trx1 m -S-AB and Trx1 m -Met-1-S-AB is the main peak, and polysulfide modified forms (-SS-AB, -SSS-AB, -SSSS-AB) account for only 20% ( Figure 4 E and 4F). The above results indicate that HPB is significantly superior to IAB in labeling and stabilizing polysulfide-modified proteins.

[0091] Application of 2.5HPB in proteomic polysulfide modification analysis

[0092] Traditional proteomic analysis of polysulfide modifications usually involves directly adding alkylating agents to cell lysates for in situ labeling (Scheme 1, Figure 5 A). However, high concentrations of small molecule thiols (such as GSH) and polysulfides (such as GSSH) in cells can consume or reduce the alkylation reagent, resulting in low labeling efficiency. To this end, we optimized the experimental process: adding a protein precipitation step after cell lysis to remove small molecule interfering substances, and then performing alkylation labeling after resolubilizing the protein (Process 2, Figure 5 A).

[0093] To verify whether the precipitation-redissolution step affects the Pr-S n H stability and labeling efficiency, HSSH modified Trx1 m After this step, the cells were labeled with HPB. n -PB content did not decrease but increased ( Figure 5 B and 5C), and the proportion increased from 44.3% to 55.4% ( Figure 5 D), proving that this step can effectively improve the enrichment efficiency of polysulfide-modified proteins. Based on this, the polysulfide modification analysis of the proteome of colon cancer cell HT29 was performed using process 2. The results showed that the amount of potential polysulfide-modified proteins captured by the HPB labeling group was significantly higher than that of the IAB group ( Figure 5 E).

[0094] Due to the presence of non-specific alkylation, the protein obtained in process 2 may be mixed with non-target modification products. Therefore, it was further optimized to process 3: after the labeled protein was cleaved by enzyme, the peptides were enriched by streptavidin magnetic beads, and the resulting peptides were divided into two categories: cysteine (specific modification) and non-cysteine (non-specific modification). Using process 3 to analyze the HT29 proteome, it was found that cysteine peptides accounted for 73.3% in the HPB labeled group, while only 67.4% in the IAB group ( Figure 5 F), indicating that HPB has a lower false positive rate.

[0095] In summary, this study successfully synthesized the polysulfide modification detection tag HPB through a four-step reaction. The tag is based on the HPE-IAM structure and is conjugated to biotin. When reacting with glutathione persulfide (GSSH), HPB only exhibited alkylation activity, and no reduced HPB was detected; in contrast, IAB showed significant redox activity, resulting in the production of reduced IAB. In peptide alkylation experiments, HPB had significantly fewer nonspecific modification sites than IAB. Ultimately, in the labeling and screening of polysulfide-modified proteins in the HT29 cell proteome, the proportion of false positive peptides with HPB was lower than that with IAB. These results indicate that compared to the commonly used alkylating agent IAB, HPB has significant advantages in reaction specificity and polysulfide chain retention.

[0096] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art will be able to modify the technical solutions described in the aforementioned embodiments or substitute equivalents for some of the technical features. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. A high-fidelity biotinylated probe, characterized in that The probe has the following structure:

2. The method for preparing a high-fidelity biotinylated probe according to claim 1, wherein The preparation method includes the following synthetic route:

3. The preparation method according to claim 2, wherein The preparation method comprises: S1: Biotin, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI), 4-dimethylaminopyridine (DMAP) and N-tert-butyloxycarbonyl-L-tyrosine were mixed to generate intermediate I, which was purified by silica gel flash column chromatography; S2: intermediate I is treated with hydrochloric acid (HCl) to remove the Boc protecting group to obtain intermediate II; S3: Intermediate II reacts with diisopropylethylamine (DIPEA) and chloroacetyl chloride to generate intermediate III containing a chlorine atom; S4: Intermediate III is reacted with potassium iodide (KI) to replace the chlorine atom with the iodine atom.

4. The preparation method according to claim 3, wherein In step S1, the molar ratio of biotin, EDCI, DMAP and N-tert-butyloxycarbonyl-L-tyrosine is 1-5:1-5:10-30:1-5; In the step S2, the molar ratio of the intermediate I to hydrochloric acid is 0.5-5:1-5; In the step S3, the molar ratio of the intermediate II, DIPEA and chloroacetyl chloride is 0.5-5:1-5:1-5; In the step S4, the molar ratio of the intermediate III to KI is 1:0.5-5.

5. A detection kit, characterized in that: The kit at least comprises the probe according to claim 1.

6. Use of the probe according to claim 1 or the detection kit according to claim 5 in the detection of small molecule polysulfides or protein polysulfide modifications.

7. The use according to claim 7, characterized in that The application is to treat small molecule polysulfides (including GS n H and Cysteine-S n H) or the labeling and screening of polysulfide-modified proteins in the cell proteome.

8. A method for detecting and analyzing polysulfide-modified proteins in a cell proteome, characterized in that: The method comprises: adding cells into a cell lysis solution containing the probe according to claim 1 or the detection kit according to claim 5 for lysis, thereby achieving alkylation labeling.

9. A method for detecting and analyzing polysulfide-modified proteins in a cell proteome, characterized in that: The method comprises: adding cells to a lysis solution and then performing protein precipitation, re-dissolving the protein and then adding the probe according to claim 1 or the detection kit according to claim 5, thereby performing alkylation labeling.

10. The method according to claim 8 or 9, characterized in that The alkylated labeled protein was enzymatically digested and then peptides were enriched using streptavidin magnetic beads. The resulting peptides were divided into two categories: those containing cysteine (specific modification) and those containing non-cysteine (non-specific modification).

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