A method for enriching O-GlcNAc glycopeptides based on reversible chemoenzymatic labeling

Abstract

The present invention discloses a method for enriching O-GlcNAc glycopeptides based on reversible chemical enzymatic labeling-hydrophilic interaction chromatography enrichment. Specifically, it relates to: chemical enzymatic labeling of O-GlcNAc glycopeptides based on the transglycosylation activity of mutants of endoglycosidases such as Endo-M, transferring the N-sugar chain with an oxazoline structure as a whole to the O-GlcNAc glycosyl, and then using hydrophilic interaction chromatography to enrich the labeled O-GlcNAc glycopeptides in one step. For the enriched O-GlcNAc glycopeptides with introduced N-sugar chain structure, the labeled sugar chain is removed by wild-type endoglycosidase to achieve traceless enrichment of O-GlcNAc glycopeptides. Compared with other O-GlcNAc glycopeptide enrichment technologies, the present invention has the advantages of high O-GlcNAc glycopeptide enrichment efficiency and simple operation, and provides a new solution and enrichment method for the study of O-GlcNAc glycoproteomics.

Description

Technical Field

[0001] The present invention belongs to the research field of post-translational modification of glycosylated proteins in the proteomics research direction, and specifically involves novel chemical enzymatic labeling, enrichment and chemical enzymatic cleavage of O-GlcNAc sugar chains, thereby achieving efficient enrichment and high-sensitivity detection of O-GlcNAc glycopeptides without mass labels. Background Art

[0002] Protein O-GlcNAc modification is a crucial post-translational modification ubiquitous in eukaryotic cells, regulating physiological processes such as gene expression, signal transduction, immune response, and energy metabolism, and interacts extensively with phosphorylation. Abnormal protein O-GlcNAc modification is closely associated with major diseases such as cancer, diabetes, cardiovascular disease, and neurodegeneration. Therefore, in-depth research on protein O-GlcNAc modification is crucial for the early diagnosis and prevention of diseases.

[0003] High-performance liquid chromatography-mass spectrometry is the most effective method for large-scale analysis of protein O-GlcNAc modification. However, the abundance of O-GlcNAc-modified proteins or peptides in complex biological samples is extremely low, and their ion signal intensity is not only suppressed by non-glycosylated peptides but also interfered with by N-linked glycans and other O-glycan-modified fragments. Therefore, highly selective enrichment of O-GlcNAc-modified proteins / peptides is key to high-coverage analysis.

[0004] Currently, there are three main methods for enriching O-GlcNAc glycopeptides or glycoproteins, including lectin affinity chromatography (Reference 1. Trinidad, JC et al., Mol. Cell. Proteomics, 2012, 11, 215-229; Reference 2. Xu, SL. et al., Proc. Natl. Acad. Sci. USA, 2017, 11, 215-229), hydrazide chemistry (Reference 3. Klement, E. et al., J. Proteome Res., 2010, 9, 2200-2206), and click chemistry to link functional groups (Reference 4. Parker, B. et al., J. Proteome Res., 2011, 10, 1449-1458; Reference 5. Tsumoto, H. et al., J. Proteome Res., 2011, 10, 1449-1458). al., Bioorg. Med. Chem. Lett., 2015, 25, 2645-2649). The principle of hydrophilic interaction chromatography is to utilize the effect of glycosylation modification on the hydrophilicity of proteins or peptides. This method has high specificity, is simple to operate, and can maintain the integrity of the glycosyl structure. It has been widely used for the separation and enrichment of N-glycopeptides and glycoproteins (Reference 6. Chen R. et al. J. Proteomics, 2014, 103, 194-203) and has also been used for the enrichment of O-GlcNAc glycopeptides (Reference 8. Shen, B. et al., Talanta 2017, 169, 195-202). However, compared with N-linked sugar chains and other O-linked sugar chain modifications, O-GlcNAc modification composed of a single sugar group does not significantly improve the hydrophilicity of proteins or peptides, thereby limiting the selectivity and efficiency of hydrophilic enrichment of O-GlcNAc glycopeptides. The method based on hydrazide chemistry mainly uses sodium periodate oxidation to produce aldehyde groups for O-GlcNAc, and then uses hydrazide chemistry for enrichment. However, it is difficult to oxidize the trans-vicinal diol hydroxyl group in the O-GlcNAc glycoform, resulting in low enrichment efficiency. At present, enzymatic chemical labeling based on Cu(I) click chemistry has the advantage of high enrichment specificity and is widely used in the enrichment and analysis of O-GlcNAc. However, it has the challenges of cumbersome labeling and enrichment steps, difficulty in achieving efficient release of enriched peptides, and large mass labels of enriched glycopeptides, resulting in low identification sensitivity of peptides. Therefore, the development of new chemical enzymatic labeling methods to improve the enrichment specificity and identification efficiency of glycopeptides has become the key to O-GlcNAc glycosylation analysis.

[0005] Previous reports indicate that the N175Q mutant of the endoglycosidase Endo-M from Mucor hiemalis essentially lost its hydrolytic activity but was able to transfer oligosaccharide chains en bloc to single N-linked GlcNAc sugars (Katoh T. et al. J. Biol. Chem., 2016, 291, 23305-23317). In the present invention, we utilize Endo-MN175Q to en bloc transfer N-glycan analogs bearing oxazoline-reactive groups to the O-GlcNAc sugars of glycopeptides. Hydrophilic interaction chromatography (HIC) is then used to achieve one-step enrichment of the labeled glycopeptides, simplifying the enrichment process and improving glycopeptide enrichment efficiency. Furthermore, addressing the issue of chemical enzymatic labeling introducing mass tags, wild-type Endo-M enzyme is used to reversibly remove the labeled sugars, thereby achieving traceless enrichment of O-GlcNAc glycopeptides and improving the sensitivity and efficiency of mass spectrometry detection, thereby facilitating the efficient enrichment and analysis of O-GlcNAc glycosylation. Summary of the Invention

[0006] The present invention aims to provide a method for efficiently, high-throughput, and highly specific analysis of O-GlcNAc-modified proteome information in complex biological samples. The method is based on the fact that the modified O-GlcNAc groups on peptides can be transferred to oxazoline-structured sugar chains CT (composed of (Gal)2(GlcNAc)3(Man)3, with a relative molecular mass of 1419.5) by the endoglycosidase Endo-MN175Q from Mucor truncatula. This allows for tight non-covalent binding to the epoxyazidemaltose hydrophilic chromatography material, thereby enriching and separating O-GlcNAc glycopeptides from complex samples. Multiple wild-type endoglycosidases are then used in combination to cleave the transferred sugar chains, restoring the O-GlcNAc groups on the peptides. The O-GlcNAc glycoproteome information of the sample is then obtained through mass spectrometry analysis. This method can be generally described as a high-throughput analysis of O-GlcNAc glycoproteome information in samples using hydrophilic interaction chromatography based on the overall transfer and cleavage of sugar chains.

[0007] The present invention adopts the following technical solutions:

[0008] First, protein samples from cells and / or tissues were extracted, digested with trypsin, and then treated with glycosidase PNGase F. The oxazoline-structured sugar chain CT (composition: (Gal)2(GlcNAc)3(Man)3, relative molecular mass 1419.5) was then transferred to the O-GlcNAc sugar group of the peptide using the endoglycosidase mutant Endo-MN175Q from Mucor truncatus. The labeled O-GlcNAc peptides were enriched using maltose hydrophilic chromatography material. The wild-type endoglycosidase Endo-M WT from Mucor truncatus and the wild-type endoglycosidase Endo-S WT from Streptococcus pyogenes were then used in combination to remove the CT sugar chain while retaining the in situ O-GlcNAc sugar group for the identification of O-GlcNAc glycosylation sites, O-GlcNAc glycopeptides, and corresponding glycoprotein information.

[0009] The specific steps include:

[0010] 1) Take biological samples and use the commercialized Minute TM SC-003 kit was used to prepare cytoplasmic protein extraction samples and nuclear protein extraction samples (operation reference Minute TM The protein concentration of the nuclear extract sample was determined using the BCA method according to the SC-003 kit instructions. Dithiothreitol (DTT) was then added to a final concentration of 10 mM. The mixture was incubated at 95°C in a water bath for 5 min. After cooling to room temperature, iodoacetamide (IAM) was added to a final concentration of 20 mM. The mixture was incubated at 20-25°C in the dark for 30 min and mixed thoroughly.

[0011] 2) adding 0.2 mg of the denatured nuclear protein sample obtained in step 1) to a 10 kDa ultrafiltration membrane washed with water, centrifuging at 14,000 × g at 20-25°C until the membrane bottom is exposed, adding 0.1 ml of an aqueous solution containing 8 M urea and 100 mM ammonium bicarbonate to each ultrafiltration membrane, centrifuging at 14,000 × g at 20-25°C until the membrane bottom is exposed, discarding the filtrate, adding 0.1 ml of an aqueous solution containing 100 mM ammonium bicarbonate to each ultrafiltration membrane, centrifuging at 14,000 × g at 20-25°C until the membrane bottom is exposed, repeating twice, and discarding the filtrate;

[0012] 3) Add 0.2 ml of an aqueous solution containing 20 mM ammonium bicarbonate and 20 μl of an aqueous solution containing 10 μg of trypsin to each ultrafiltration membrane in step 2), seal the membrane, and place it in a shaker at 37°C for 16 hours for enzyme digestion. Centrifuge at 14,000 × g at 20-25°C until the membrane bottom is exposed. Add 50 μl of an aqueous solution containing 20 mM ammonium bicarbonate to each ultrafiltration membrane, centrifuge at 14,000 × g at 20-25°C until the membrane bottom is exposed. Repeat this twice, and add PNGase to the filtrate at a mass ratio of 1 unit / μg to the peptide. F, placed in a shaker at 37°C for 16 h, after which trifluoroacetic acid (TFA) was added to a final concentration of 1%, mixed, and then used with a commercial C18 solid-phase extraction column purchased from Waters Corporation in the United States to remove other small molecules. The peptide fragments in 1 ml of the collected eluate (composed of 80% acetonitrile (ACN) / 0.1% formic acid (FA) aqueous solution, v / v) were freeze-dried to 0.2 mg / portion;

[0013] 4) Dissolve the dried peptide obtained in step 3) in 200 mM sodium phosphate buffer (pH 7.2), centrifuge at 14,000 × g at 20-25°C, take the supernatant, add CT-ox (stock concentration of 70 mM, relative molecular mass 1419.5) to a final concentration of 9 mM and mix well. Then add Endo-M N175Q (stock concentration of 20 mg / ml) to a final concentration of 0.45 mg / ml and mix well to make the final peptide concentration of 7 mg / ml. Incubate in a shaker at 37°C for 2 h. After the reaction is complete, add TFA to a final concentration of 1% and mix well. Add a commercial C18 solid-phase extraction column purchased from Waters Corporation to remove other small molecules. The resulting 0.5 ml peptide eluate (80% ACN / 0.1% FA aqueous solution, v / v) is freeze-dried and washed with 200 μl of 80% ACN / 1% Dissolve the mixture in TFA aqueous solution (v / v), centrifuge at 20,000×g at 20-25°C, take the supernatant and add it to 5 mg of washed epoxyazide maltose hydrophilic chromatography material, shake at 1200 rpm at 25°C for 1 hour, then add 1 mm thick C18 material (material particle size 5 μm, membrane pore size 1.5 μm) for retention on the membrane, remove the filtrate, wash the material with 150 μl 80% ACN / 1% TFA aqueous solution (v / v), discard the filtrate, repeat twice, and finally add 250 μl 30% ACN / 1% formic acid (FA) aqueous solution (v / v) to the material for elution, collect the filtrate and freeze-dry;

[0014] 5) Dissolve the dried peptide obtained in step 4) in 30 μl of 50 mM sodium phosphate buffer, add Endo-M WT (see Umekawa M. et al., 2010, J. Biol. Chem. pp. 512–513) to a final concentration of 0.2 mg / ml and Endo-S WT (see Huang W. et al., 2012, J. Am. Chem. Soc. p. 12316) to a final concentration of 0.5 mg / ml, and incubate in a shaker at 37°C for 12 h. After digestion, add TFA to a final concentration of 1% and mix thoroughly. Add a commercial C18 solid-phase extraction column purchased from Waters Corporation to remove other small molecules. The resulting 150 μl peptide eluate (80% ACN / 0.1% FA aqueous solution, v / v) was freeze-dried.

[0015] 6) Dissolve the filtered fraction from step 5) in 200 μl of 80% ACN / 1% TFA (v / v) aqueous solution, centrifuge at 20,000 × g at 20-25°C, add the supernatant to 5 mg of washed maltose epoxide azide hydrophilic chromatography material, shake at 1200 rpm at 25°C for 1 h, and then add a 1 mm thick C18 membrane (material particle size 5 μm, membrane pore size 1.5 μm) for retention. Then, wash the material with 150 μl of 80% ACN / 1% TFA (v / v) aqueous solution, collect the filtrate, repeat twice, combine with the first filtrate, and freeze-dry. Finally, add 250 μl of 30% ACN / 1% FA (v / v) aqueous solution to elute the material, collect the filtrate, and freeze-dry.

[0016] 7) The combined freeze-dried sample of the first three filtrates obtained in step 6) was dissolved in 100 μl of 10 mM ammonium bicarbonate aqueous solution B (pH 10.0), and 2 mg of commercial Durashell C18 material chromatographic column (available from Agela, USA) was added. The column was eluted with 100 μl of each wash solution of ACN and 10 mM ammonium bicarbonate aqueous solution B (pH 10.0) at 12 different mixing ratios. The filtrates were collected and combined into 6 fractions. Each fraction was freeze-dried and dissolved in 15 μl of 1% FA aqueous solution (v / v). The supernatant was centrifuged at 25,000 × g for 3 min to obtain the O-GlcNAc glycopeptide sample. The sample was then directly analyzed by MALDI-TOF / TOF MS or LC-MS / MS to obtain information on O-glycosylation sites, O-glycopeptides, and corresponding glycoproteins in the sample.

[0017] The pH of the aqueous solution containing 8M urea and 100mM ammonium bicarbonate in step 2) is 7.5-8.0; the pH of the aqueous solution containing 100mM ammonium bicarbonate in step 2) is 7.5-8.0; the pH of the aqueous solution containing 20mM ammonium bicarbonate in step 3) is 7.5-8.0.

[0018] The 200 mM sodium phosphate buffer described in step 4) is formulated as a mixture of 28 ml of a 200 mM NaH2PO4 aqueous solution and 72 ml of a 200 mM Na2HPO4 aqueous solution, with a pH of 7.2. The 50 mM sodium phosphate buffer described in step 5) is formulated as a mixture of 2.8 ml of a 50 mM Na2HPO4 aqueous solution and 7.2 ml of a 50 mM NaH2PO4 aqueous solution, with a pH of 6.3 to 6.5. The 10 mM ammonium bicarbonate aqueous solution B described in step 7) is formulated as a 100 mM ammonium bicarbonate aqueous solution at a pH of 10.0 diluted to 10 mM with ultrapure water (the experimental water was obtained using a Milli-Q water treatment system from Millipore Corporation, USA, the same below).

[0019] The washing of the ultrafiltration membrane with water in steps 2) and 5) is specifically performed by adding 0.2 ml of ultrapure water to a 10 kDa ultrafiltration membrane purchased from Sartorius, Germany, centrifuging at 14,000 × g for 20 min at 20-25° C., and discarding the filtrate; the washing of the epoxy azide maltose material in steps 4) and 6) is specifically performed by ultrasonically cleaning with 0.3 ml of 80% ACN / 1% TFA aqueous solution (v / v) for 6 min, then centrifuging at 14,000 × g at 20-25° C., discarding the supernatant, and repeating this three times; the removal of small molecules from the solution using a C18 solid phase extraction column in step 3) is specifically performed by adding 6 column volumes of 0.1% TFA aqueous solution (v / v) to the extraction column that has been added with the sample and naturally drained for elution.

[0020] The homemade 2 mg Durashell C18 column described in step 7) was prepared by ultrasonically cleaning 2 mg of Durashell C18 material with 0.2 ml of a 50% ACN / 50% ammonium bicarbonate aqueous solution B (v / v) mixture for 5 minutes. A 1 mm thick C18 material (5 μm particle size, 1.5 μm membrane pore size) in a 200 μl tip was then added. The liquid was drained from the membrane, and the material was then washed once with 0.2 ml of an 80% ACN / 20% ammonium bicarbonate aqueous solution B (v / v) mixture, 0.2 ml of ACN, and 0.2 ml of a 10 mM ammonium bicarbonate aqueous solution B, respectively.

[0021] The sample was fractionated into 6 fractions as described in step 7), and the specific operation was as follows: (1) 0.1 ml of a 3% ACN / 97% ammonium bicarbonate aqueous solution B (v / v) mixture, (2) 0.1 ml of a 6% ACN / 94% ammonium bicarbonate aqueous solution B (v / v) mixture, (3) 0.1 ml of a 9% ACN / 91% ammonium bicarbonate aqueous solution B (v / v) mixture, (4) 0.1 ml of a 12% ACN / 88% ammonium bicarbonate aqueous solution B (v / v) mixture, (5) 0.1 ml of a 15% ACN / 85% ammonium bicarbonate aqueous solution B (v / v) mixture, (6) 0.1 ml of a 18% ACN / 82% ammonium bicarbonate aqueous solution B (v / v) mixture, (7) 0.1 ml of a 21% ACN / 85% ammonium bicarbonate aqueous solution B (v / v) mixture, 0.1 ml of ACN / 79% aqueous ammonium bicarbonate solution B (v / v) mixture, (8) 0.1 ml of 25% ACN / 75% aqueous ammonium bicarbonate solution B (v / v) mixture, (9) 0.1 ml of 30% ACN / 70% aqueous ammonium bicarbonate solution B (v / v) mixture, (10) 0.1 ml of 35% ACN / 65% aqueous ammonium bicarbonate solution B (v / v) mixture, (11) 0.1 ml of 50% ACN / 50% aqueous ammonium bicarbonate solution B (v / v) mixture, after each addition, the filtrate was discharged and collected, and then the filtrate of (1) was combined with the filtrate of (7), (2) was combined with the filtrate of (8), (3) was combined with the filtrate of (9), (4) was combined with the filtrate of (10), (5) was combined with the filtrate of (11), and the filtrate of (6) was combined with the filtrate of the first step. The 6 tubes of samples obtained were freeze-dried separately.

[0022] This method can simultaneously obtain the identification results of the corresponding O-GlcNAc glycoproteins, O-GlcNAc glycopeptides and O-GlcNAc glycosylation sites, and can be used for proteomic analysis of O-GlcNAc modification.

[0023] Labeling of O-GlcNAc glycopeptides:

[0024] (1) The labeling enzyme uses the N175Q mutant of the endoglycosidase Endo-M. The endoglycosidase has the activity of hydrolyzing sugar chains. The use of its specific mutant can effectively reduce its hydrolysis activity and enhance its activity of transferring sugar chains to the O-GlcNAc group on the peptide segment, thereby being used for the specific labeling of O-GlcNAc glycopeptides;

[0025] (2) The sugar unit is labeled with an N-linked sugar analog of an oxazoline structure, and based on the sugar chain transfer activity of the enzyme described in (1), the similar structure of the N-linked sugar chain is transferred to the O-GlcNAc sugar group;

[0026] (3) The labeling target is O-GlcNAc glycopeptide / glycoprotein. Based on the recognition ability of endoglycosidase mutants for GlcNAc glycosyl groups and the tolerance for peptide sequences, the O-GlcNAc glycosyl groups in glycopeptides and glycoproteins are selectively labeled;

[0027] Enrichment of O-GlcNAc glycopeptides after labeling:

[0028] (1) Hydrophilic interaction chromatography enrichment of O-GlcNAc glycopeptides. After chemical enzymatic labeling, the sugar chain has a structure similar to that of N-linked sugar chains and exhibits strong hydrophilicity. With the help of non-covalent bond forces such as hydrophilic interaction chromatography, O-GlcNAc glycopeptides can be enriched in one step.

[0029] (2) The enrichment target of hydrophilic interaction chromatography is O-GlcNAc-labeled glycopeptides. Hydrophilic interaction chromatography has been widely used to enrich N-linked intact glycopeptides. The present invention is the first to use hydrophilic interaction chromatography to enrich labeled O-GlcNAc glycopeptides.

[0030] Sugar chain release of O-GlcNAc glycopeptides after enrichment: Based on the recognition specificity of wild-type endoglycosidase for glycoform structures and its tolerance for peptide sequences and connection modes, wild-type endoglycosidase is used to remove the labeled sugar chains introduced during the enrichment of O-GlcNAc glycopeptides, thereby restoring the monosaccharide structure on the O-GlcNAc glycopeptide. This avoids the challenges of low fragmentation efficiency and spectral retrieval faced in the glycopeptide detection process, thereby improving the sensitivity and coverage of O-GlcNAc glycopeptide detection.

[0031] This method enriches N-glycan-containing O-GlcNAc glycopeptides and uses an endoglycosidase to remove the labeled sugar chains, allowing for traceless enrichment of O-GlcNAc glycopeptides. Compared to other O-GlcNAc glycopeptide enrichment techniques, this method offers advantages such as high O-GlcNAc glycopeptide enrichment efficiency and ease of operation, providing a new solution and enrichment method for O-GlcNAc glycoproteomics research.

[0032] The hydrophilic interaction chromatography method based on overall sugar chain transfer is to transfer and enrich the O-GlcNAc glycopeptides in the sample at the polypeptide level after trypsin hydrolysis.

[0033] The hydrophilic interaction chromatography method based on the overall transfer of sugar chains can be carried out as follows (eg Figure 1As shown): the sample to be analyzed is digested into peptides with a 20:1 mass ratio of trypsin, and then the N-glycan chains are removed with the glycosidase PNGaseF. Small molecules including N-glycan chains are removed with a commercial C18 solid phase extraction column. Then, the N175Q mutant of the endoglycosidase Endo-M is used to transfer the CT sugar group of the oxazoline structure (composition: (Gal)2(GlcNAc)3(Man)3, relative molecular mass: 1419.5) to the O-GlcNAc group of the polypeptide. The O-GlcNAc glycopeptide with the transferred sugar chain is then enriched using epoxyazide maltose hydrophilic chromatography material in an 80% ACN / 1% TFA aqueous solution. The CT sugar chain of the enriched product is removed by combining two wild-type endoglycosidases, Endo-MWT and Endo-S WT. Finally, the product is purified by a homemade Durashell The C18 column was used to fractionate the peptides with restored O-GlcNAc sugar groups and used for mass spectrometry analysis to obtain the O-GlcNAc glycoproteome information in the sample.

[0034] The hydrophilic interaction chromatography method based on overall sugar chain transfer, after removing the interference of N-sugar chains, transfers CT sugar chains to the O-GlcNAc group of the polypeptide to enhance the hydrophilicity of the O-GlcNAc glycopeptide, thereby improving the efficiency and specificity of the epoxyazide maltose hydrophilic chromatography material in enriching O-GlcNAc glycopeptides, increasing the coverage of the O-GlcNAc glycosylated proteome, and reducing the interference of non-glycosylated proteins.

[0035] Advantages of the present invention:

[0036] The method described in the present invention has obvious advantages: high efficiency, high throughput, and high specificity. Hydrophilic interaction chromatography has been widely used in glycoproteomics analysis, but there are few reports on its use in enriching O-GlcNAc glycopeptides. Epoxyazide maltose hydrophilic chromatography material has the advantages of easy preparation, easy separation, and high enrichment specificity. The experimental process of the present invention is to apply hydrophilic interaction chromatography at the polypeptide level, which has high universality in shotgun proteomics; the present invention uses the overall transfer of sugar chains for the first time to achieve the labeling and enrichment separation of O-GlcNAc glycopeptides in the sample, combined with high-resolution RPLC-MS / MS analysis, to improve the identification coverage of the O-GlcNAc glycosylated proteome and reduce the interference of non-glycosylated proteins, thereby obtaining the identification results of O-GlcNAc-modified glycoproteins, glycopeptides and glycosylation sites. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments:

[0038] Figure 1 The figure is an experimental flow chart of the hydrophilic interaction chromatography method based on the overall transfer of sugar chains.

[0039] Figure 2 MALDI-TOF mass spectra demonstrating the feasibility of the hydrophilic interaction chromatography (HIC) method based on the overall transfer of glycan chains. The sample was a 1:1000 mass ratio mixture of a standard O-GlcNAc glycopeptide and a tryptic digest of a standard non-glycoprotein, BSA. a) Direct analysis of the mixture; b) HIC analysis based on the overall transfer of glycan chains.

[0040] Figure 3 Comparison of the results of hydrophilic interaction chromatography (HIC) based on the overall transfer of glycans and direct HIC for the identification of O-GlcNAc glycosylation sites in nuclear proteins from Thiamet-G-treated HeLa cells. a) Identification results from two replicates of the HIC method; b) Identification results from two replicates of the direct HIC method; c) Comparison of the overall identification results of the two methods. DETAILED DESCRIPTION

[0041] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0042] The following materials and reagents were used in the examples:

[0043] Standard O-GlcNAc glycopeptides were purchased from Nanjing Jiepeptide Co., Ltd. Bovine serum albumin (BSA), 1,4-dithiothreitol (DTT), iodoacetamide (IAM), trypsin, and 2,5-dihydroxybenzoic acid (DHB) were purchased from Sigma (IL, USA). Peptide-N-glycosidase F (PNGaseF) was purchased from New England Biolabs (MA, USA). Water used in the experiments was purified using a Milli-Q water system purchased from Millipore (MA, USA). All other reagents were of analytical grade or higher.

[0044] Example 1

[0045] Verify the feasibility of the hydrophilic interaction chromatography method based on the overall transfer of sugar chains:

[0046] Bovine serum albumin (BSA) is a commonly used standard non-glycoprotein. The hydrophilic interaction chromatography method based on the overall transfer of sugar chains described in the present invention was used to enrich and separate standard O-GlcNAc glycopeptides from a mixture. The experimental process is as follows:

[0047] 1. Weigh 1 mg of standard O-GlcNAc glycopeptide, dissolve in 1 ml of Milli-Q ultrapure water, and store at -80°C.

[0048] 2. Dissolve 2 mg of BSA in 0.5 ml of an aqueous solution containing 8 M urea and 100 mM ammonium bicarbonate (Note: This operation is to simulate the enzymatic hydrolysis of biological samples and dissolve standard non-glycoproteins). Add dithiothreitol (DTT) to a final concentration of 10 mM and react in a 37°C water bath for 2 h to open the disulfide bonds. After cooling to 20-25°C, add iodoacetamide (IAM) to a final concentration of 20 mM and react in the dark for 30 min to block the sulfhydryl groups. Divide into 0.2 mg portions and add Insert the sample into 10 10KDa German Sartorius ultrafiltration membranes and centrifuge at 14,000×g at 20-25°C. Add 0.1ml of an aqueous solution containing 8M urea and 100mM ammonium bicarbonate to each membrane and centrifuge at 14,000×g at 20-25°C. Discard the filtrate. Add 0.1ml of an aqueous solution containing 100mM ammonium bicarbonate to the ultrafiltration membrane for washing and centrifuge at 14,000×g at 20-25°C. Repeat the washing process twice and discard the filtrate.

[0049] 3. Add 0.2 ml of an aqueous solution containing 20 mM ammonium bicarbonate and 20 μl of an aqueous solution containing 10 μg of trypsin to the ultrafiltration membrane from step 2. Place the membrane in a shaker at 37°C for 16 h, centrifuge at 14,000 × g at 20-25°C, then add 45-55 μl of an aqueous solution containing 18-22 mM ammonium bicarbonate to the ultrafiltration membrane for washing. Centrifuge at 14,000 × g at 20-25°C, and repeat the washing process 1-2 times. (Note: Since BSA is not a glycoprotein, PNGase F was not added in this experiment.) The filtrates were combined and directly added to a final concentration of 1% trifluoroacetic acid (TFA). After mixing, other small molecules were removed using a C18 solid phase extraction column from Waters, USA. The peptide fragments collected in 1 ml of the eluate (composed of 80% acetonitrile (ACN) / 0.1% formic acid (FA) aqueous solution, v / v) were freeze-dried to 0.2 mg / portion.

[0050] 4. Dissolve 0.2 mg of the dried sample in 30 μl of 200 mM sodium phosphate buffer (pH 7.2) (the final peptide concentration is approximately 6.7 μg / μl) and add 0.2 μl of the 1 mg / ml standard O-GlcNAc glycopeptide aqueous solution prepared in step 1 and mix thoroughly.

[0051] 5. The obtained mixed peptide sample was centrifuged at 14000×g at 20-25°C. The supernatant was collected and mixed with oxazolidinone glycosyl donor CT-ox (composition: (Gal)2(GlcNAc)3(Man)3, stock solution concentration: 70mM, relative molecular mass: 1419.5; purchased from Shanghai Institute of Materia Medica, Chinese Academy of Sciences, prepared by dehydration from egg yolk, method: Huang W. et al., 2009, J. Am. Chem. Soc. p. 2222) at a final concentration of 9mM. Endo-M N175Q (stock solution concentration: 20mg / ml, purchased from Shanghai Institute of Materia Medica, Chinese Academy of Sciences, preparation method: Umekawa M. et al., 2009, J. Am. Chem. Soc. p. 2222) at a final concentration of 0.45mg / ml. et al., 2010, J.Biol.Chem.pp.512–513), and the mixture was stirred at 37°C for 2 h. After the reaction, TFA was added to a final concentration of 1% and mixed. Small molecules were removed using a C18 solid-phase extraction column from Waters, USA. The column was eluted with 1 ml of 80% acetonitrile (ACN) / 0.1% formic acid (FA) aqueous solution (v / v) and collected. After freeze-drying, the column was dissolved in 200 μl of 80% ACN / 1% TFA aqueous solution (v / v). The column was centrifuged at 14,000 × g at 25°C, and the supernatant was added with 5 mg of washed epoxyazide maltose hydrophilic chromatography material (prepared by the Dalian Institute of Chemical Physics, Chinese Academy of Sciences, according to the method of Yu L. et al., 2010, J.Biol.Chem.pp.512–513). et al., 2009, Chem. Eur. J. pp. 12618–12626), shaken at 1200 rpm for 1 h at 25°C, then added to a 1 mm thick C18 material (material particle size 5 μm, membrane pore size 1.5 μm) membrane for retention, discarding the filtrate, and then washing the material with 150 μl 80% ACN / 1% TFA aqueous solution (v / v), discarding the filtrate, repeating the washing twice, and finally adding 250 μl 30% ACN / 1% FA aqueous solution (v / v) to elute the material, and collecting the filtrate for freeze-drying;

[0052] 6. The obtained product was dissolved in 30 μl of 50 mM sodium phosphate buffer (pH 6.4), and wild-type Endo-M WT (purchased from Shanghai Institute of Materia Medica, Chinese Academy of Sciences, for preparation method, see Umekawa M. et al., 2010, J. Biol. Chem. pp. 512–513) at a final concentration of 0.2 mg / ml and wild-type Endo-S WT (purchased from Shanghai Institute of Materia Medica, Chinese Academy of Sciences, for preparation method, see Huang W. et al., 2012, J. Am. Chem. Soc. p. 12316) at a final concentration of 0.5 mg / ml were added. The mixture was incubated at 37°C for 12 h for enzyme digestion to obtain enriched O-GlcNAc glycopeptide samples, which were then directly analyzed by MALDI-TOF-MS.

[0053] Note: Since the protein sample used in this experiment is not a biological sample and the glycopeptide used is only one standard glycopeptide, there is no need to perform the fractionation operation in step 7) of the technical solution in the manual.

[0054] Analysis results:

[0055] like Figure 2 As shown in b), the final product spectrum after the above steps has only one in situ standard O-GlcNAc glycopeptide peak (NNLEES*(GlcNAc)LLKLE), and its relative intensity is more than 5 times that of the strongest impurity peak. The results of direct analysis of 1 / 400 of the BSA peptide-standard O-GlcNAc glycopeptide mixed sample obtained in step 4 of this example are as follows: Figure 2 As shown in a), there are multiple peptides in the spectrum, but no peak of standard O-GlcNAc glycopeptide can be seen. Obviously, the hydrophilic interaction chromatography based on overall sugar chain transfer can highly selectively enrich O-GlcNAc glycopeptide from a complex peptide mixed sample.

[0056] Example 2

[0057] We used the aforementioned hydrophilic interaction chromatography based on overall sugar chain transfer and direct hydrophilic interaction chromatography to analyze the O-GlcNAc glycoproteome information in nuclear proteins of HeLa cells treated with Thiamet-G, and compared the results of O-GlcNAc glycosylation sites identified by the two methods.

[0058] The HeLa cells used in this experiment were cultured for 24 hours with 5 μM Thiamet-G (purchased from Selleck, USA). The experimental procedure for applying hydrophilic interaction chromatography (HIIC) based on the overall transfer of sugar chains to nuclear samples is as follows:

[0059] 1. Take HeLa cells and use the commercialized Minute TM SC-003 kit was used to prepare cytoplasmic protein extraction samples and nuclear protein extraction samples (operation reference Minute TM The protein concentration of the nuclear extract sample was determined by the BCA method according to the SC-003 kit instructions. Dithiothreitol (DTT) was then added to a final concentration of 10 mM and the mixture was incubated in a 37°C water bath for 2 h. After cooling to room temperature, iodoacetamide (IAM) was added to a final concentration of 20 mM and the mixture was reacted at 20-25°C in the dark for 30 min and mixed thoroughly.

[0060] 2. Add 0.2 mg of denatured nuclear protein sample to a 10 kDa ultrafiltration membrane purchased from Sartorius, Germany, after washing with water. Centrifuge at 14,000 × g at 25°C until the membrane bottom is exposed. Add 0.1 ml of an aqueous solution containing 8 M urea and 100 mM ammonium bicarbonate to each ultrafiltration membrane. Centrifuge at 14,000 × g at 25°C until the membrane bottom is exposed. Discard the filtrate. Next, wash each ultrafiltration membrane by adding 0.1 ml of an aqueous solution containing 100 mM ammonium bicarbonate. Centrifuge at 14,000 × g at 25°C until the membrane bottom is exposed. Repeat the washing process twice, and discard the filtrate.

[0061] 3. Add 0.2 ml of an aqueous solution containing 20 mM ammonium bicarbonate and 20 μl of an aqueous solution containing 10 μg of trypsin to each ultrafiltration membrane. After sealing, enzymatic digestion was carried out on a shaker at 37°C for 16 h. Centrifuge at 14,000 × g at 25°C until the membrane bottom was exposed. Add 50 μl of an aqueous solution containing 20 mM ammonium bicarbonate to each ultrafiltration membrane and centrifuge at 14,000 × g at 25°C until the membrane bottom was exposed. Repeat twice. Add PNGase F purchased from New England Biolabs, USA, at a mass ratio of 1 unit / μg to the peptide to the filtrate. Enzymatic digestion was carried out on a shaker at 37°C for 16 h. Trifluoroacetic acid (TFA) was then added to a final concentration of 1%. After mixing, other small molecules were removed using a C18 solid phase extraction column purchased from Waters, USA. The peptides eluted with 1 ml of eluent (80% ACN / 0.1% formic acid (FA) aqueous solution, v / v) were freeze-dried to 0.2 mg / portion.

[0062] 4. Dissolve 0.2 mg of the obtained dried sample in 30 μl of 200 mM PB buffer (pH 7.2) and centrifuge at 14,000 × g at 25°C. Remove the supernatant and add CT-ox (composition: (Gal)2(GlcNAc)3(Man)3, stock solution concentration: 70 mM, relative molecular mass: 1419.5 g / mol, prepared from egg yolk dehydration at the Shanghai Institute of Materia Medica, Chinese Academy of Sciences; method: Huang W. et al., 2009, J. Am. Chem. Soc., p. 2222) to a final concentration of 9 mM, and mix thoroughly. Then add Endo-M N175Q (stock solution concentration: 20 mg / ml, prepared at the Shanghai Institute of Materia Medica, Chinese Academy of Sciences; method: Umekawa M. et al., 2009, J. Am. Chem. Soc., p. 2222) to a final concentration of 0.45 mg / ml. et al., 2010, J.Biol.Chem.pp.512–513) and mixed, placed in a shaker at 37°C for 2 h. After the reaction was completed, TFA was added to a final concentration of 1% and mixed. Small molecules were removed using a C18 solid-phase extraction column from Waters, USA. The peptides were eluted with 1 ml of eluent (80% ACN / 0.1% formic acid (FA) aqueous solution, v / v). After lyophilization, 200 μl of 80% ACN / 1% TFA aqueous solution (v / v) was added for dissolution. The mixture was centrifuged at 20,000 × g at 25°C. The supernatant was added with 5 mg of washed epoxyazide maltose hydrophilic chromatography material (prepared by the Dalian Institute of Chemical Physics, Chinese Academy of Sciences, according to the method of Yu L. et al., 2010, J.Biol.Chem.pp.512–513). et al., 2009, Chem. Eur. J. pp. 12618–12626), shaken at 1200 rpm for 1 h at 25°C, then transferred to a 1 mm thick C18 membrane (material particle size 5 μm, membrane pore size 1.5 μm) for retention, discarding the filtrate, and then washing the material with 150 μl 80% ACN / 1% TFA aqueous solution (v / v), discarding the filtrate, repeating the washing twice, and finally adding 250 μl 30% ACN / 1% FA aqueous solution (v / v) to elute the material, and collecting the filtrate for freeze-drying;

[0063] 5. The resulting product was dissolved in 30 μl of 50 mM sodium phosphate buffer (pH 6.4), and wild-type Endo-M WT (prepared by the Shanghai Institute of Materia Medica, Chinese Academy of Sciences, for methods see Umekawa M. et al., 2010, J. Biol. Chem. pp. 512–513) and wild-type Endo-S WT (prepared by the Shanghai Institute of Materia Medica, Chinese Academy of Sciences, for methods see Huang W. et al., 2012, J. Am. Chem. Soc. p. 12316) were added to a final concentration of 0.2 mg / ml. The product was incubated for 12 h at 37°C in a shaker. After digestion, the product was mixed with a final concentration of 1% TFA. Other small molecules were removed using a commercial C18 solid-phase extraction column purchased from Waters Corporation, USA. The peptides were eluted with 1 ml of eluent (80% ACN / 0.1% formic acid (FA) aqueous solution, v / v) and freeze-dried.

[0064] 6. The eluted fractions from step 5) were dissolved in 200 μl of 80% ACN / 1% TFA aqueous solution (v / v), centrifuged at 20,000 × g at 20-25°C, and the supernatant was added to 5 mg of washed epoxyazide maltose material (prepared by the Dalian Institute of Chemical Physics, Chinese Academy of Sciences, for the method see Yu L. et al., 2009, Chem. Eur. J. pp. 12618–12626). After shaking at 1200 rpm for 1 h at 25°C, the supernatant was added to a 1 mm thick C18 material (material particle size 5 μm, membrane pore size 1.5 μm) membrane for retention. The filtrate was collected and then washed with 150 μl of 80% ACN / 1% TFA aqueous solution (v / v). The resulting filtrate was combined with the first step filtrate and washed twice. The three filtrate fractions were combined and freeze-dried to obtain the O-GlcNAc glycopeptide sample, which was then directly added to 15 μl of The solution was reconstituted with 0.1% FA aqueous solution and then analyzed by LC-MS / MS.

[0065] Note: The experimental subjects in this example were biological samples from HeLa cells. The purpose of this experiment was to evaluate and compare the hydrophilic interaction chromatography method based on the transfer of sugar chains described in the present invention with direct hydrophilic interaction chromatography (specific process: the dried peptide fragments of HeLa cell nuclei obtained in step 3 of this example were directly dissolved in 200 μl of 80% ACN / 1% TFA aqueous solution (v / v), centrifuged at 20,000 × g at 25°C, and the supernatant was added to 5 mg of washed epoxyazide maltose hydrophilic chromatography material. The supernatant was shaken at 1200 rpm at 25°C for 1 h, and then transferred to a 1 mm thick C18 material (material particle size 5 μm, membrane pore size 1.5 μm) membrane for retention. The filtrate was discarded, and the material was then washed with 150 μl of 80% ACN / 1% TFA aqueous solution (v / v). The filtrate was discarded, and the washing was repeated twice. Finally, 250 μl of 30% ACN / 1% The material was eluted with aqueous FA solution (v / v), and the filtrate was collected and freeze-dried. Performance of enrichment of O-GlcNAc glycopeptides from equal amounts of HeLa cell nuclear peptides.

[0066] Analysis results:

[0067] We used the above hydrophilic interaction chromatography based on the overall transfer of sugar chains and direct hydrophilic interaction chromatography to perform three technical replicates on the same amount of starting samples. Figure 3 The results given in a) and 3b) are the results of three technical repetitions. It can be seen that the hydrophilic interaction chromatography based on the overall transfer of sugar chains identified a total of 657 unique O-GlcNAc glycosylation sites (corresponding to 187 glycoproteins) through the three technical repetitions, while the direct hydrophilic interaction chromatography method only identified 25 unique O-GlcNAc glycosylation sites (corresponding to 16 glycoproteins) after the same experiment (for the specific process, see Yu L. et al., 2009, Chem. Eur. J. pp. 12618–12626). This shows that the hydrophilic interaction chromatography based on the overall transfer of sugar chains significantly improves the identification coverage of O-GlcNAc glycosylation sites in nuclear proteins. Figure 3 As shown in Figure c), after comparing the O-GlcNAc glycosylation sites identified by the two methods, it was found that all 25 sites identified by direct hydrophilic interaction chromatography were also identified by hydrophilic interaction chromatography based on integrated sugar chain transfer, demonstrating the superiority of the hydrophilic interaction chromatography based on integrated sugar chain transfer described in the present invention.

[0068] in conclusion

[0069] In summary, the present invention has developed a method for enriching O-GlcNAc glycopeptides based on reversible chemoenzymatic labeling-hydrophilic interaction chromatography enrichment, which simplifies the enrichment process and improves the enrichment efficiency of glycopeptides. On this basis, to address the problem of introducing mass tags during chemoenzymatic labeling, wild-type endoglycosidases Endo-M WT and Endo-S WT are used to remove the labeled glycans, completing the traceless enrichment of O-GlcNAc glycopeptides and improving their mass spectrometry detection sensitivity and efficiency, thereby achieving efficient enrichment and analysis of O-GlcNAc glycosylation. The present invention provides a new solution and enrichment method for the study of O-GlcNAc glycoproteomics.

Claims

1. A method for reversible chemoenzymatic labeling and enrichment of O-GlcNAc glycopeptides, characterized by: This method uses the N175Q mutant of endoglycosidase Endo-M to transfer oligosaccharide chains to O-GlcNAc glycosyl groups to increase hydrophilicity, and then uses maltose hydrophilic interaction chromatography material to enrich O-GlcNAc extended glycopeptides. For the enriched sugar chains, wild-type endoglycosidases Endo-M WT and Endo-S WT were used to remove the labeled sugar chains to obtain O-GlcNAc glycopeptides in the biological sample. The specific steps are as follows: (1) Chemoenzymatic labeling of O-GlcNAc glycopeptides. The specific operation is as follows: using oxazoline-structured sugar chain CT as the labeling reagent, the oxazoline-structured sugar chain CT composition is (Gal)2(GlcNAc)3(Man)3, the final concentration is 8-10 mM, the relative molecular mass is 1419.5, the preparation method is described in Huang W. et al., 2009, J. Am. Chem. Soc. p. 2222, and the N175Q mutant of endoglycosidase Endo-M is used at a final concentration of 0.4-0.5 μg / μl in 180-220 mM sodium phosphate buffer at pH 7.1-7.3 at 36-38°C to catalyze the labeling of sugar chains to the O-GlcNAc group in the biological peptide sample; (2) Enrichment of O-GlcNAc-labeled glycopeptides: Based on the strong hydrophilicity of extended O-GlcNAc-labeled glycopeptides, the selective enrichment of O-GlcNAc-labeled glycopeptides was achieved by utilizing the non-covalent bond of the hydrophilic interaction chromatography material of epoxyazidemaltose in a 78-82% acetonitrile (ACN) / 0.9-1.1% trifluoroacetic acid (TFA) system at room temperature; (3) O-GlcNAc glycopeptide sugar chain excision: Based on the enzymatic activity and specificity of endoglycosidase, the labeled sugar chain was excised using wild-type endoglycosidase Endo-M WT at a concentration of 0.18-0.22 μg / μl and Endo-S WT at a final concentration of 0.45-0.55 μg / μl in 45-55 mM sodium phosphate buffer at pH 6.3-6.5 at 36-38°C to restore the structure of the O-GlcNAc sugar group.

2. The method according to claim 1, wherein The biological sample refers to one or more tissues and / or cells of animals, plants, or microorganisms; The process of obtaining biological peptide samples is as follows: extract protein samples from cells or tissues, hydrolyze them with trypsin, and then treat them with glycosidase PNGase F to obtain biological peptide samples.

3. The method according to claim 2, wherein: Enrichment of O-GlcNAc glycopeptides in tissues and cells; A reversible chemoenzymatic labeling and enrichment method for O-GlcNAc glycopeptides assisted by endoglycosidase mutants is applied to the analysis and detection of O-GlcNAc glycosylation in biological samples, thereby obtaining the identification results of O-GlcNAc-modified glycoproteins, glycopeptides, and glycosylation sites, which can be used to study the regulatory mechanism of glycosylation of key proteins in tumor metabolism.

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