CN114149479A - Preparation method of solid-phase enriched O-GlcNAc glycopeptide - Google Patents
Preparation method of solid-phase enriched O-GlcNAc glycopeptide Download PDFInfo
- Publication number
- CN114149479A CN114149479A CN202111388738.6A CN202111388738A CN114149479A CN 114149479 A CN114149479 A CN 114149479A CN 202111388738 A CN202111388738 A CN 202111388738A CN 114149479 A CN114149479 A CN 114149479A
- Authority
- CN
- China
- Prior art keywords
- glycopeptide
- glcnac
- solid phase
- polypeptide
- glycan
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
- C07K1/1072—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups
- C07K1/1077—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups by covalent attachment of residues other than amino acids or peptide residues, e.g. sugars, polyols, fatty acids
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/04—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length on carriers
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/12—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by hydrolysis, i.e. solvolysis in general
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/145—Extraction; Separation; Purification by extraction or solubilisation
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/36—Extraction; Separation; Purification by a combination of two or more processes of different types
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/005—Glycopeptides, glycoproteins
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
Abstract
The application relates to a preparation method of solid-phase enriched O-GlcNAc glycopeptide, which comprises the following steps: s1, providing a polypeptide mixture and a solid phase material, wherein the polypeptide mixture comprises glycan-free polypeptide, N-glycan polypeptide, mucin type O-glycopeptide and O-GlcNAc glycopeptide; s21, oxidizing the polypeptide mixture, covalently bonding the polypeptide mixture to a solid phase material, and removing N-glycan to obtain solid phase-bonded O-glycopeptide and O-GlcNAc glycopeptide; or S22, removing N-glycan of the polypeptide mixture, and then binding the polypeptide mixture on a solid phase material through affinity to obtain O-glycopeptide and O-GlcNAc glycopeptide bound by a solid phase; and S3, further carrying out enzyme digestion or hydrolysis to obtain the O-GlcNAc glycopeptide. The enriched O-GlcNAc glycopeptide and the site specificity of the O-glycopeptide prepared by the method are both in a higher level, so that the difficult problems of enriched O-GlcNAc glycopeptide and low site specificity in the O-glycopeptide prepared by the prior art are solved.
Description
Technical Field
The invention relates to a preparation method of solid-phase enriched O-GlcNAc glycopeptide, belonging to the technical field of biomolecule preparation and analysis.
Background
O-GlcNAcylation is a nutrient and stress responsive post-translational modification (PTM) involving the attachment of O-N-acetylglucosamine (O-GlcNAc) to Ser and Thr residues of cytoplasmic, nuclear and mitochondrial proteins. A pair of enzymes, O-GlcNAc transferase (OGT) and O-GlcNAcase (O-GlcNA glycosidase, OGA), which add and remove O-GlcNAc controls the dynamic cycle of the PTM. O-GlcNAcylation is involved in the spatio-temporal regulation of a variety of cellular processes, including transcription, epigenetic modification, and cellular signaling kinetics. O-GlcNAcylation is highly dynamic, usually transient, but the underlying mechanism of O-GlcNAcylation signal timing is largely unknown. O-GlcNAcylation can be considered as an essential "oil and glue" for cells: acting as a "grease" by coating the protein of interest (folded or unfolded, mature or new) and preventing unwanted protein aggregation or modification. While also acting as a "glue" that affects the function of various proteins in the cell by modulating protein-protein interactions in time and space in response to internal and external cues.
An increase in overall O-GlcNAcylation is a common feature of cancer cells. Recent studies have shown that O-GlcNAcylation is a central propagator of the nutritional state and can control key signaling and metabolic pathways that regulate multiple cancer cell phenotypes. In addition, EMT-HBP plays a role in improving the level of protein O-GlcNAcylation, and the occurrence and the progression of KRAS mutation-induced lung tumors are accelerated. A common method of studying O-GlcNAcylation is by antibody assay or click chemistry. However, these methods lack specificity, and therefore, it is important to establish a novel technique for identifying an O-GlcNAc substrate for studying the function of an O-GlcNAc protein in cancer.
Disclosure of Invention
The invention aims to provide a preparation method of a solid-phase enriched O-GlcNAc glycopeptide, which solves the problems of enriched O-GlcNAc glycopeptide and low site specificity in the O-glycopeptide prepared in the prior art.
In order to achieve the purpose, the invention provides the following technical scheme: a preparation method of solid-phase enriched O-GlcNAc glycopeptide, which comprises the following steps:
s1, providing a polypeptide mixture and a solid phase material, wherein the polypeptide mixture comprises glycan-free polypeptide, N-glycan polypeptide, mucin type O-glycopeptide and O-GlcNAc glycopeptide;
s21, oxidizing the polypeptide mixture, covalently bonding the polypeptide mixture to the solid phase material, and removing N-glycan to obtain solid-phase-bonded O-glycopeptide and O-GlcNAc glycopeptide;
alternatively, the first and second electrodes may be,
s22, removing N-glycan of the polypeptide mixture, and then binding the polypeptide mixture on the solid phase material through affinity to obtain solid-phase-bound O-glycopeptide and O-GlcNAc glycopeptide;
and the number of the first and second groups,
and S3, further carrying out enzyme digestion or hydrolysis to obtain the O-GlcNAc glycopeptide.
Further, said oxidizing said polypeptide mixture and removing N-glycans by covalently binding to said solid phase material to obtain a solid phase bound O-glycopeptide and O-GlcNAc glycopeptide comprising:
oxidizing glycan on glycopeptide in the polypeptide mixture into oxidized glycopeptide with aldehyde group by an oxidant, extracting the oxidized glycopeptide, and covalently bonding the oxidized glycopeptide with a solid phase material with hydrazide or amino on the surface;
and adding N-glycosidase into the mixture to carry out enzymolysis on the N-glycan on the solid phase to obtain O-glycopeptide and O-GlcNAc glycopeptide combined with the solid phase.
Further, after removing the N-glycans of the polypeptide mixture, affinity-binding the polypeptide mixture to the solid phase material to obtain a solid-phase-bound O-glycopeptide and an O-GlcNAc glycopeptide, comprising:
adding N-glycosidase into the polypeptide mixture, carrying out enzyme digestion to remove N-glycan, obtaining a mixed solution containing glycan-free polypeptide, mucin-type O-glycopeptide, O-GlcNAc glycopeptide and N-glycan, and then removing the N-glycan through purification to obtain a sample solution;
dissolving the sample solution in a lectin buffer solution and adding the lectin buffer solution to a lectin resin, and carrying out affinity binding on the O-glycopeptide with an O-GlcNAc structure and the lectin resin to obtain the solid-phase-bound O-GlcNAc glycopeptide.
Further, the S3 includes:
adding O-GlcNAc glycosidase into the O-glycopeptide and O-GlcNAc glycopeptide combined with the solid phase, carrying out enzymolysis on the O-GlcNAc glycopeptide from serine or threonine, collecting supernatant, and purifying to obtain the O-GlcNAc-removed polypeptide with an O-GlcNAc locus.
Further, the S3 includes:
and adding an acidic solution into the O-glycopeptide and the O-GlcNAc glycopeptide bonded with the solid phase, hydrolyzing a hydrazone bond formed by the O-GlcNAc glycopeptide and the hydrazide solid phase material, and collecting supernatant to obtain the O-glycopeptide with an O-GlcNAc site and oxidized O-GlcNAc.
Further, the step of adding an acidic solution to the solid-phase bound O-GlcNAc glycopeptide further comprises adjusting the pH of the mixture after the addition of the acidic solution to less than 3.
Further, the solid phase material is a resin.
Further, the S3 includes:
adding O-GlcNAc glycosidase dissolved in MES prepared with heavy water to the solid phase-bound O-glycopeptide and O-GlcNAc glycopeptide, performing enzymolysis on the O-GlcNAc glycopeptide, and analyzing the components by a bioinformatics method.
Further, the polypeptide mixture is derived from tissue cells, which are benign and/or cancer tissue cells.
Further, still include:
s0, respectively taking 5-10 mg benign and cancer tissues, freezing the tissues, then smashing the tissues by using a tissue homogenizer, and adding lysate and a proteolysis inhibitor into the smashed tissues.
Compared with the prior art, the invention has the beneficial effects that: the preparation method of the solid-phase enriched O-glycopeptide firstly extracts protein, then uses protease to hydrolyze to obtain polypeptide, then covalently enriches the purified polypeptide or enriches the polypeptide on the solid phase in an affinity manner, uses N-glycosidase to remove the polypeptide on the N-glycopeptide on the solid phase, the solid phase is reserved as the O-glycopeptide and the O-GlcNAc glycopeptide, and the enriched O-GlcNAc glycopeptide and the site specificity are both in a higher level.
Meanwhile, the O-glycopeptide is subjected to enzymolysis through O-GlcNAcase glycosidase, so that the O-GlcNAc glycopeptide site and the sequence thereof can be determined. The method has important significance for researching the discovery and the function of the O-GlcNAcylation protein substrate in cancer, diabetes, degenerative nerve and the like.
The foregoing description is only an overview of the technical solutions of the present invention, and in order to make the technical solutions of the present invention more clearly understood and to implement them in accordance with the contents of the description, the following detailed description is given with reference to the preferred embodiments of the present invention and the accompanying drawings.
Drawings
FIG. 1 is a flow diagram of a method for the preparation of a solid phase enriched O-GlcNAc glycopeptide of the present application;
FIG. 2 is a schematic diagram showing the steps of a method for preparing a solid-phase enriched O-GlcNAc glycopeptide according to the first embodiment of the present application;
FIG. 3 is a schematic diagram showing the steps of the method for producing a solid-phase enriched O-GlcNAc glycopeptide according to example II of the present application;
FIG. 4 is a schematic diagram showing the steps of determining the O-GlcNAc site by digestion of OGA according to the third embodiment of the present application;
FIG. 5 is a schematic diagram showing the steps of determining the O-GlcNAc site by acidic hydrolysis according to example four of the present application;
FIG. 6 is a schematic diagram showing the steps for determining the O-GlcNAc glycosylation site according to example five of the present application;
fig. 7 is a schematic step diagram of an application of the preparation method of the sixth embodiment of the present application in a human body.
Detailed Description
The following detailed description of embodiments of the present invention is provided in connection with the accompanying drawings and examples. The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.
It should be noted that: the terms "upper", "lower", "left", "right", "inner" and "outer" of the present invention are used for describing the present invention with reference to the drawings, and are not intended to be limiting terms.
Referring to fig. 1, the present application discloses a method for preparing a solid-phase enriched O-GlcNAc glycopeptide, which comprises the following steps:
s1, providing a polypeptide mixture and a solid phase material, wherein the polypeptide mixture comprises glycan-free polypeptide, N-glycan polypeptide, mucin type O-glycopeptide and O-GlcNAc glycopeptide;
s21, oxidizing the polypeptide mixture, covalently bonding the polypeptide mixture to the solid phase material, and removing N-glycan to obtain solid-phase-bonded O-glycopeptide and O-GlcNAc glycopeptide;
alternatively, the first and second electrodes may be,
s22, removing N-glycan of the polypeptide mixture, and then binding the polypeptide mixture on the solid phase material through affinity to obtain solid-phase-bound O-glycopeptide and O-GlcNAc glycopeptide;
and the number of the first and second groups,
and S3, further carrying out enzyme digestion or hydrolysis to obtain the O-GlcNAc glycopeptide.
Specifically, in S21, the oxidizing the polypeptide mixture and covalently binding to the solid phase material to remove N-glycans to obtain solid-phase bound O-glycopeptide and O-GlcNAc glycopeptide, includes:
oxidizing glycan on glycopeptide in the polypeptide mixture into oxidized glycopeptide with aldehyde group by an oxidant, extracting the oxidized glycopeptide, and covalently bonding the oxidized glycopeptide with a solid phase material with hydrazide or amino on the surface;
and adding N-glycosidase into the mixture to carry out enzymolysis on the N-glycan on the solid phase to obtain O-glycopeptide and O-GlcNAc glycopeptide combined with the solid phase.
Specifically, in S22, after removing N-glycans of the polypeptide mixture, affinity-binding the polypeptide mixture to the solid phase material to obtain a solid-phase-bound O-glycopeptide and O-GlcNAc glycopeptide, including:
adding N-glycosidase into the polypeptide mixture, carrying out enzyme digestion to remove N-glycan, obtaining a mixed solution containing glycan-free polypeptide, mucin-type O-glycopeptide, O-GlcNAc glycopeptide and N-glycan, and then removing the N-glycan through purification to obtain a sample solution;
dissolving the sample solution in a lectin buffer solution and adding the lectin buffer solution to a lectin resin, and carrying out affinity binding on the O-glycopeptide with an O-GlcNAc structure and the lectin resin to obtain the solid-phase-bound O-GlcNAc glycopeptide.
Optionally, S3 includes:
adding O-GlcNAc glycosidase into the O-glycopeptide combined with the solid phase, carrying out enzymolysis on the O-GlcNAc glycopeptide from serine or threonine, collecting supernatant, and purifying to obtain the O-GlcNAc-removed polypeptide with an O-GlcNAc locus.
Optionally, S3 includes:
and adding an acidic solution into the O-glycopeptide combined with the solid phase, hydrolyzing a hydrazone bond formed by the O-GlcNAc glycopeptide and the hydrazide solid phase material, and collecting supernatant to obtain the O-glycopeptide with an O-GlcNAc site and oxidized O-GlcNAc. Wherein, the step of adding the acidic solution into the O-glycopeptide bound to the solid phase further comprises the step of ensuring that the pH of the mixed solution after the acidic solution is added is less than 3.
Optionally, the solid phase material is a resin.
Optionally, S3 further includes:
adding O-GlcNAc glycosidase dissolved in MES prepared with heavy water to the solid phase-bound O-glycopeptide, hydrolyzing the O-GlcNAc glycopeptide, and analyzing the components by bioinformatics. Wherein said polypeptide mixture is derived from tissue cells, said tissue cells being benign and/or cancer tissue cells.
Optionally, the method further includes:
s0, respectively taking 5-10 mg benign and cancer tissues, freezing the tissues, then smashing the tissues by using a tissue homogenizer, and adding lysate and a proteolysis inhibitor into the smashed tissues.
The present application will be further described with reference to specific examples.
EXAMPLES A covalent conjugation preparation of solid phase conjugated O-glycopeptides
Please refer to fig. 2, in detail:
freezing the tissue in dry ice or-80 deg.C for 2-3 hr, taking out, and immediately crushing the tissue;
adding 400-;
taking 2-4 mul samples, diluting by 5-10 times, and testing the concentration of the protein by using BCA;
according to the concentration, 1000 mu g of protein is dissolved in 600 mu l of urea with the total volume of 400-600 mu l and the final concentration of 8M, and the sample is slightly oscillated to ensure that the protein is completely dissolved;
adding 80-100 μ l 120mM Disulfitol (DTT), and reacting at 37 deg.C for 1-1.5 hr;
adding 80-100 μ l 160mM iodoacetamide, and reacting in dark room at room temperature for 1-1.5 hr;
diluting the sample by 5-6 times, adding newly prepared 1M ammonium bicarbonate to obtain final ammonium bicarbonate concentration of 20-25mM, and testing the pH value of the sample to be 7-9;
adding 20-25 μ g sequencing-grade trypsin, slightly oscillating, reacting at 37 deg.C for 16-18 hr, and hydrolyzing to obtain polypeptide;
trifluoroacetic acid (TFA, > 99%, w/v) (about 10-20. mu.l) was added to the solution until the pH was adjusted to 2-3;
pretreating the C18 extraction column, adding the sample, adding the filtrate into the extraction column, washing the extraction column with 0.1% TFA for 5-6 times (1.0-1.2ml), eluting polypeptide with 400 μ l and 50% Acetonitrile (ACN) containing 0.1% TFA, and repeating the last step for 2 times;
combining the washed polypeptides, and carrying out vacuum freeze drying to obtain purified polypeptides, wherein the purified polypeptides mainly comprise polysaccharide-free polypeptides, N-glycan polypeptides, mucin type O-glycopeptide and O-GlcNAc glycopeptide;
re-dissolving the polypeptide in 0.1% TFA and 50% ACN solution, adding 10-20mM oxidant sodium periodate, reacting at 37 deg.C for 1-2 hr, and oxidizing N-glycan, mucin-type O-glycan and O-GlcNAc on glycopeptide to form glycopeptide with aldehyde group;
after oxidation of the glycans, the glycopeptides were lyophilized in vacuo and redissolved in 1ml of 0.1% TFA, purified by C18, and then eluted from C18 using 0.1% TFA and 50% ACN;
100 μ l of spherical resin with hydrazide or amino group on the surface was added into a 1.5-2.0ml centrifuge tube. Pretreating the resin, namely washing twice with 400-;
mixing 1-2 μ l PNGaseF with 1 time Tris buffer solution, adding into resin, reacting at 37 deg.C for 2-4 hr to enzymolyze N-glycan, while retaining O-glycopeptide on solid phase;
and washing and purifying the O-glycopeptide bound on the solid phase by a liquid phase to obtain the solid-phase bound glycopeptide.
EXAMPLE two affinity binding preparation of solid phase bound O-glycopeptides
Please refer to fig. 3, in detail:
vacuum freeze-drying 800-1000 μ g of polypeptide purified by C18, and re-dissolving in 400-600 μ l of PBS buffer solution at pH 7.2-7.6;
adding 1-2 units of N-glycosidase (PNGaseF) into the sample, reacting for 4-6 hours at 37 ℃ to remove N-glycan enzyme digestion, and obtaining glycan-free polypeptide, mucin-type O-glycopeptide, O-GlcNAc glycopeptide and N-glycan in the solution;
adding 20-30 μ l concentrated TFA (> 99%, w/v) to the sample, lowering the pH to below 3.0, purifying with C18 to remove N-glycan, and vacuum freeze-drying the purified sample;
the purified sample was re-dissolved in 400-; (ii) a
Washing 200-;
adding 400-500 mu l of polypeptide sample into the pre-treated WGA resin, oscillating and mixing uniformly, and reacting overnight at room temperature;
an O-glycopeptide having an O-GlcNAc structure is affinity bound to WGA resin while other glycopeptide or non-glycopeptide polypeptides remain in the supernatant, thereby separating the O-glycopeptide from the non-glycopeptide;
the lectin resin was washed 4-5 times with 400-500. mu.l WGA buffer to obtain solid phase bound O-GlcNAc glycopeptides.
Example determination of O-GlcNAc sites by TriOGA enzymatic digestion
Please refer to fig. 4, in detail:
the O-glycopeptides covalently bound to the solid phase include O-GlcNAc glycopeptides and mucin-type O-glycopeptides, N-glycopeptides having been cleaved by PNGaseF, or
The O-glycopeptide which is affinity bonded on the solid phase is mainly O-GlcNAc glycopeptide;
adding 2-4 units of O-GlcNAc glycosidase, OGA, 200-;
hydrogen (H) on water molecules in the reaction solution is combined with oxygen (O) of serine (serine) or threonine (threonine) to form complete amino acid;
the supernatant was collected by centrifugation (1500RPM, 1-2 min) and washed 2-3 times with 400. mu.l of 0.1% TFA in deionized water, all supernatants were combined and purified by C18 to give polypeptides with O-GlcNAc sites but no O-GlcNAc monosaccharides, which were analyzed directly by mass HCD sequencing.
EXAMPLE Tetraacidic conditional hydrolysis to determine O-GlcNAc sites
Please refer to fig. 5, in detail:
the O-glycopeptides bound to the solid phase include O-GlcNAc glycopeptides and mucin-type O-glycopeptides;
the method is suitable for non-reduced hydrazone, and can be hydrolyzed under acidic conditions;
400 μ l of 0.1% TFA acid solution, or other acid solution, was added to the resin sample to ensure a pH below 3, and the reaction was carried out at room temperature for 8-12 hours. Reversibly hydrolyzing a hydrazone bond formed by the O-GlcNAc glycopeptide and the hydrazide solid phase material, and re-entering the supernatant;
centrifuging the resin (1500RPM, 1-2 min) to collect the supernatant, repeating this step 2-3 times, combining all supernatants, purifying with C18, and vacuum freeze drying to obtain O-GlcNAc glycopeptide;
this sample has O-GlcNAc sites and monosaccharides, and the O-GlcNAc is an oxidized structure, decreasing the molecular weight by 2Da (losing two hydrogens).
EXAMPLE pentaOGA enzymatic hydrolysis followed by determination of O-GlcNAc glycosylation sites by deuterium oxide
Please refer to fig. 6, in detail:
oxidized or lectin-bound O-glycopeptides, with O-GlcNAc bound to the solid phase at one end. Wherein the oxidized end forms hydrazone (hydrazone) with the resin and lectin WGA is affinity binding;
the O-GlcNAc is connected with the serine or the threonine through-O-Ser/Thr, and the specific structure is Ser/Thr-O-GlcNAc;
2-4 units of O-GlcNAc glycosidase, OGA, 200-300. mu.l of 50mM MES and 100mM NaCl, all reagents were dissolved in heavy water, mixed with the resin sample and reacted at 37 ℃ for 6-8 hours;
combining the heavy water D with O-GlcNAc glycopeptide-O-Ser/Thr to form DO-Ser/Thr, wherein Ser or Thr without O-sugar site is HO-Ser/Thr;
the difference between the molecular weight of DO-Ser/Thr and the molecular weight of HO-Ser/Thr is 1Da, and the mass spectrum analysis sets Ser/Thr as dynamic modification and increases the molecular weight by 1 Da.
EXAMPLE VI application of the preparation method in human body
Please refer to fig. 7, which specifically:
respectively taking 5-10 mg of tissues (benign/normal and cancer), freezing the sample at-80 ℃ for 2-4 hours or putting the sample into liquid nitrogen for 2-3 hours, taking out the sample, and crushing the tissue by using a tissue homogenizer;
adding 400-600 microliter of lysate and 4-6 microliter of proteolysis inhibitor into the smashed tissue, and extracting the protein according to the mode of the application;
o-glycoproteins differentially expressed by O-glcnanimation in benign/normal and cancer proteins;
using an SPGlcE method to fix, enrich and enzymolyze to obtain O-GlcNAc polypeptide;
the biological function of O-GlcNAc in cancer and the protein substrate of O-GlcNAcylation were analyzed by bioinformatics.
In summary, the following steps: the preparation method of the solid-phase enriched O-glycopeptide firstly extracts protein, then uses protease to hydrolyze to obtain polypeptide, then covalently enriches the purified polypeptide or enriches the polypeptide on the solid phase in an affinity manner, uses N-glycosidase to remove the polypeptide on the N-glycopeptide on the solid phase, the solid phase is reserved as the O-glycopeptide, and the enriched O-GlcNAc substrate and the site specificity of the prepared O-glycopeptide are both in a higher level.
Meanwhile, the O-glycopeptide is subjected to enzymolysis through O-GlcNAcase glycosidase, so that the O-GlcNAc glycopeptide site and the sequence thereof can be determined. The method has important significance for researching the discovery and the function of the O-GlcNAcylation protein substrate in cancer, diabetes, degenerative nerve and the like.
The technical features of the embodiments may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments are not described, but should be considered as the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Claims (10)
1. A preparation method of solid-phase enriched O-GlcNAc glycopeptide is characterized by comprising the following steps:
s1, providing a polypeptide mixture and a solid phase material, wherein the polypeptide mixture comprises glycan-free polypeptide, N-glycan polypeptide, mucin type O-glycopeptide and O-GlcNAc glycopeptide;
s21, oxidizing the polypeptide mixture, covalently bonding the polypeptide mixture to the solid phase material, and removing N-glycan to obtain solid-phase-bonded O-glycopeptide and O-GlcNAc glycopeptide;
alternatively, the first and second electrodes may be,
s22, removing N-glycan of the polypeptide mixture, and then binding the polypeptide mixture on the solid phase material through affinity to obtain solid-phase-bound O-glycopeptide and O-GlcNAc glycopeptide;
and the number of the first and second groups,
and S3, further carrying out enzyme digestion or hydrolysis to obtain the O-GlcNAc glycopeptide.
2. The method of claim 1, wherein said oxidizing a mixture of polypeptides and covalently attaching said oxidized polypeptide mixture to said solid phase material to remove N-glycans to provide a solid phase bound O-glycopeptide and O-GlcNAc glycopeptide, comprising:
oxidizing glycan on glycopeptide in the polypeptide mixture into oxidized glycopeptide with aldehyde group by an oxidant, extracting the oxidized glycopeptide, and covalently bonding the oxidized glycopeptide with a solid phase material with hydrazide or amino on the surface;
and adding N-glycosidase into the mixture to carry out enzymolysis on the N-glycan on the solid phase to obtain O-glycopeptide and O-GlcNAc glycopeptide combined with the solid phase.
3. The method of claim 1, wherein said removing N-glycans from said polypeptide mixture and affinity binding said polypeptide mixture to said solid phase material provides a solid phase bound O-glycopeptide and O-GlcNAc glycopeptide, comprising:
adding N-glycosidase into the polypeptide mixture, carrying out enzyme digestion to remove N-glycan, obtaining a mixed solution containing glycan-free polypeptide, mucin-type O-glycopeptide, O-GlcNAc glycopeptide and N-glycan, and then removing the N-glycan through purification to obtain a sample solution;
dissolving the sample solution in a lectin buffer solution and adding the lectin buffer solution to a lectin resin, and carrying out affinity binding on the O-glycopeptide with an O-GlcNAc structure and the lectin resin to obtain the solid-phase-bound O-GlcNAc glycopeptide.
4. The method for preparing a solid-phase enriched O-GlcNAc glycopeptide of claim 2 or 3, wherein S3 comprises:
adding O-GlcNAc glycosidase into the O-glycopeptide and O-GlcNAc glycopeptide combined with the solid phase, carrying out enzymolysis on the O-GlcNAc glycopeptide from serine or threonine, collecting supernatant, and purifying to obtain the O-GlcNAc-removed polypeptide with an O-GlcNAc locus.
5. The method for preparing a solid-phase enriched O-GlcNAc glycopeptide of claim 2, wherein S3 comprises:
and adding an acidic solution into the O-glycopeptide and the O-GlcNAc glycopeptide bonded with the solid phase, hydrolyzing a hydrazone bond formed by the O-GlcNAc glycopeptide and serine or threonine, and collecting supernatant to obtain the O-glycopeptide with an O-GlcNAc site and oxidized O-GlcNAc.
6. The method of claim 5, wherein said adding an acidic solution to said solid phase-bound O-GlcNAc glycopeptide further comprises adjusting the pH of the mixture after said adding an acidic solution to less than 3.
7. The method of claim 1, wherein the solid phase material is a resin.
8. The method for preparing a solid-phase enriched O-GlcNAc glycopeptide of claim 1, wherein S3 comprises:
adding O-GlcNAc glycosidase dissolved in MES (2-morpholinoethanesulfonic acid) prepared in heavy water to the solid phase-bound O-glycopeptide and O-GlcNAc glycopeptide, hydrolyzing the O-GlcNAc glycopeptide, and analyzing the components by a bioinformatics method.
9. The method of claim 1, wherein said polypeptide mixture is derived from tissue cells, and wherein said tissue cells are benign and/or cancerous.
10. The method of claim 9, further comprising:
s0, respectively taking 5-10 mg benign and cancer tissues, freezing the tissues, then smashing the tissues by using a tissue homogenizer, and adding lysate and a proteolysis inhibitor into the smashed tissues.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111388738.6A CN114149479A (en) | 2021-11-22 | 2021-11-22 | Preparation method of solid-phase enriched O-GlcNAc glycopeptide |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111388738.6A CN114149479A (en) | 2021-11-22 | 2021-11-22 | Preparation method of solid-phase enriched O-GlcNAc glycopeptide |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN114149479A true CN114149479A (en) | 2022-03-08 |
Family
ID=80457187
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111388738.6A Pending CN114149479A (en) | 2021-11-22 | 2021-11-22 | Preparation method of solid-phase enriched O-GlcNAc glycopeptide |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114149479A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114717224A (en) * | 2022-04-08 | 2022-07-08 | 苏州大学 | Method for enriching and identifying glycosylated ribonucleic acid (glycoRNA) based on solid phase |
| WO2023193382A1 (en) * | 2022-04-06 | 2023-10-12 | 苏州大学 | Solid-phase glycoprotein-based t antigen glycopeptide enrichment and enzymatic cleavage analysis method |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110749736A (en) * | 2018-07-24 | 2020-02-04 | 中国科学院大连化学物理研究所 | O-GalNAc glycosylated peptide segment identification method based on chemical reaction simplification |
| CN112578061A (en) * | 2020-12-15 | 2021-03-30 | 南京谱利健生物技术有限公司 | Method for separating N-glycopeptide and O-glycopeptide based on solid phase method |
| CN113058577A (en) * | 2021-03-17 | 2021-07-02 | 苏州大学 | Glycopeptide enrichment material and preparation method and enrichment method thereof |
-
2021
- 2021-11-22 CN CN202111388738.6A patent/CN114149479A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110749736A (en) * | 2018-07-24 | 2020-02-04 | 中国科学院大连化学物理研究所 | O-GalNAc glycosylated peptide segment identification method based on chemical reaction simplification |
| CN112578061A (en) * | 2020-12-15 | 2021-03-30 | 南京谱利健生物技术有限公司 | Method for separating N-glycopeptide and O-glycopeptide based on solid phase method |
| CN113058577A (en) * | 2021-03-17 | 2021-07-02 | 苏州大学 | Glycopeptide enrichment material and preparation method and enrichment method thereof |
Non-Patent Citations (3)
| Title |
|---|
| 包慧敏等: "糖蛋白质组学中基于化学反应的富集方法研究进展", 色谱, vol. 34, no. 12, pages 1145 * |
| 薛鹏等: "凝集素法与酰肼法富集糖肽的方法学比较", 生物化学与生物物理进展, vol. 42, no. 4, pages 356 - 364 * |
| 覃培斌等: "O-GlcNAc糖蛋白/糖肽的分离富集方法", 生命的化学, vol. 33, no. 2, pages 59 - 66 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2023193382A1 (en) * | 2022-04-06 | 2023-10-12 | 苏州大学 | Solid-phase glycoprotein-based t antigen glycopeptide enrichment and enzymatic cleavage analysis method |
| CN114717224A (en) * | 2022-04-08 | 2022-07-08 | 苏州大学 | Method for enriching and identifying glycosylated ribonucleic acid (glycoRNA) based on solid phase |
| CN114717224B (en) * | 2022-04-08 | 2024-01-30 | 苏州大学 | Method for enriching and identifying glycosylated ribonucleic acid glycoRNA based on solid phase |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN114149479A (en) | Preparation method of solid-phase enriched O-GlcNAc glycopeptide | |
| CN107525842B (en) | Analytical method for studying protein structure or protein interaction | |
| Tang et al. | Concanavalin A‐immobilized magnetic nanoparticles for selective enrichment of glycoproteins and application to glycoproteomics in hepatocelluar carcinoma cell line | |
| WO2023093133A1 (en) | Analysis method based on solid-phase glycoprotein enrichment and tn glycopeptide enzyme digestion, and application | |
| AU2003249692A1 (en) | Methods for quantitative proteome analysis of glycoproteins | |
| Partridge et al. | The chemistry of connective tissues. 4. The presence of a non-collagenous protein in cartilage | |
| Chen et al. | Development of a combined chemical and enzymatic approach for the mass spectrometric identification and quantification of aberrant N-glycosylation | |
| CN108333263A (en) | A kind of detection method of Urine proteins preparation method and urine protein group | |
| CN108359704A (en) | The processing method of goat dairy lactalbumin for proteomics research | |
| Bellei et al. | Proteomic analysis of early urinary biomarkers of renal changes in type 2 diabetic patients | |
| CN109342743B (en) | Preparation method of denatured IgG capable of being efficiently combined with rheumatoid factor | |
| CN111777696B (en) | Method for specific reversible enrichment of nascent protein | |
| Ramachandran et al. | Comparison of N-linked glycoproteins in human whole saliva, parotid, submandibular, and sublingual glandular secretions identified using hydrazide chemistry and mass spectrometry | |
| WO2023193382A1 (en) | Solid-phase glycoprotein-based t antigen glycopeptide enrichment and enzymatic cleavage analysis method | |
| WO2023103437A1 (en) | Method for analyzing tn antigen based on combination of solid-phase enrichment and o-glycopeptide enzymatic cleavage | |
| CN110618229B (en) | Non-reducing peptide map analysis method of protein | |
| JP5239319B2 (en) | Method for selectively recovering protein C-terminal peptide and method for determining amino acid sequence of protein C-terminal peptide using the same | |
| Kim et al. | Lectin precipitation using phytohemagglutinin‐L4 coupled to avidin–agarose for serological biomarker discovery in colorectal cancer | |
| CN103710368B (en) | The Human kallikrein of mammalian cell expression and encoding gene and application thereof | |
| CN106568875B (en) | Material for enriching sialylglycopeptide and application thereof | |
| CN102336828A (en) | Multiple myeloma specific protein and its special detection kit | |
| KR100661931B1 (en) | A method for the diagnosis of cancers by measuring the changes of glycosylation of transferrin and the diagnosis kit using the same | |
| Wang et al. | Multi-histidine functionalized material for the specific enrichment of sialylated glycopeptides | |
| CN112285265B (en) | Novel method for protein methylation modification reverse enrichment based on mirror image enzyme orthogonality principle and application | |
| KR101530210B1 (en) | Marker for gastric cancer diagnosis using N-glycopeptide |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |