Identification of Glycosylated Peptides Using Hydrophilic Nanocomposites Combined with Mass Spectrometry

Abstract

The invention belongs to the field of nanotechnology, and in particular relates to a method for identifying a glycosylation peptide fragment by combing a hydrophilic nano-composite material with mass spectrometric analysis. The method comprises the following steps: preparing dispersing liquid from a nano-composite material, taking ultrapure water as a solvent, mixing the dispersing liquid and a glycosylation peptide fragment solution in a 80 percent acetonitrile / 0.1 percent formic acid buffer solution and performing incubation in an enzymolysis instrument; magnetically separating the nano-composite material under the action of an external magnetic field, and washing with the 80 percent acetonitrile / 0.1 percent formic acid buffer solution; eluting the glycosylation peptide fragment enriched on the material with a 50 percent acetonitrile / 0.1 percent formic acid buffer solution and performing identification by combining mass spectrometric analysis. The hydrophilic nano-composite material is a nano-composite material prepared by coating the surface of magnetic graphene with poly(diallyldimethylammonium chloride) and nanogold particles and modifying glucose. The method is simple in operation, low in cost, sensitive, rapid, high in sensitivity, high in selectivity and very suitable for detection of the glycosylation peptide fragment in complex biological samples.

Description

technical field

[0001] The invention belongs to the field of nanotechnology, in particular to a method for identifying glycosylated peptides by combining hydrophilic nanocomposite materials with mass spectrometry analysis, in particular to a magnetic graphene surface-coated poly(diallyl dimethyl chloride) A method for the enrichment and detection of glycosylated peptides using a composite nanomaterial of ammonium chloride) and gold nanoparticles and modified glucose combined with mass spectrometry. Background technique

[0002] Protein glycosylation is one of the most important post-translational modifications in the life process, and plays an important role in molecular recognition, intracellular and intercellular signal transduction, immune response and other life activities. According to the records and predictions of the Swiss-Prot database, more than half of human proteins are glycosylated. The composition and abundance of glycoproteins are closely related to diseases,...

Examples

Embodiment 1

[0036] Embodiment 1: The synthesis of a hydrophilic nanocomposite material coated with poly(diallyldimethylammonium chloride) and nano-gold particles and modified glucose on the surface of a magnetic graphene

[0037] (1) Acidify graphene with concentrated nitric acid, disperse 400mg graphene in 40mL concentrated nitric acid, react at 60°C for 7 hours to obtain acidified graphene, and then fully wash acidified graphene with deionized water until the washing liquid is Until neutral, vacuum dry at 50°C;

[0038] (2) Add 150 mg of the acidified graphene obtained in step (1) to 40 mL of ethylene glycol, add 405 mg of ferric chloride hexahydrate, ultrasonically disperse for 1 hour, then add 0.15 g of trisodium citrate, 1.8 g of sodium acetate and 1.0 g of Polyethylene glycol, stirred at room temperature for 0.5 hours, then transferred the mixture to a polytetrafluoroethylene-lined stainless steel reactor, and reacted at 200°C for 12 hours to obtain magnetic graphene, and the produc...

Embodiment 2

[0047] Embodiment 2: The magnetic graphene surface that obtains in embodiment 1 coats poly(diallyl dimethyl ammonium chloride) and nano-gold particles and the hydrophilic composite nanomaterial that modifies glucose is applied to low-concentration horseradish Enrichment of oxidase HRP hydrolyzate and detection by MALDI-TOF MS

[0048] (1) Prepare standard proteolysis solution: Accurately weigh 1 mg HRP standard protein, prepare a standard protein solution with a concentration of 5 mg / mL with 25 mM ammonium bicarbonate solution, and boil for ten minutes; add dithiothreitol to make the two The final concentration of thiothreitol was 5 mM, reacted at 60°C for 1 hour, then added iodoacetamide to make the final concentration of iodoacetamide 12.5mM, and reacted for 1 hour at 37°C in the dark; then diluted with 25mM ammonium bicarbonate solution to make the final HRP The concentration is 1mg / mL, according to the ratio of trypsin and standard protein with a mass ratio of 1:40, add tr...

Embodiment 3

[0054] Embodiment 3: The magnetic graphene surface that obtains in embodiment 1 coats poly(diallyl dimethyl ammonium chloride) and nano-gold particles and the hydrophilic composite nanomaterial that modifies glucose as solid-phase microextraction adsorption separation The medium is used for the enrichment and MALDI-TOF MS detection of the mixed solution of HRP enzymolysis solution and bovine serum albumin (BSA) enzymolysis solution

[0055] (1) Prepare standard proteolysis solution: Accurately weigh 1 mg HRP standard protein, prepare a standard protein solution with a concentration of 5 mg / mL with 25 mM ammonium bicarbonate solution, and boil for ten minutes; add dithiothreitol to make the two The final concentration of thiothreitol was 5 mM, reacted at 60°C for 1 hour, then added iodoacetamide to make the final concentration of iodoacetamide 12.5mM, and reacted for 1 hour at 37°C in the dark; then diluted with 25mM ammonium bicarbonate solution to make the final HRP The conce...

technical field

[0001] The invention belongs to the field of nanotechnology, in particular to a method for identifying glycosylated peptides by combining hydrophilic nanocomposite materials with mass spectrometry analysis, in particular to a magnetic graphene surface-coated poly(diallyl dimethyl chloride) A method for the enrichment and detection of glycosylated peptides using a composite nanomaterial of ammonium chloride) and gold nanoparticles and modified glucose combined with mass spectrometry. Background technique

[0002] Protein glycosylation is one of the most important post-translational modifications in the life process, and plays an important role in molecular recognition, intracellular and intercellular signal transduction, immune response and other life activities. According to the records and predictions of the Swiss-Prot database, more than half of human proteins are glycosylated. The composition and abundance of glycoproteins are closely related to diseases,...

Examples

Embodiment 1

[0036] Embodiment 1: The synthesis of a hydrophilic nanocomposite material coated with poly(diallyldimethylammonium chloride) and nano-gold particles and modified glucose on the surface of a magnetic graphene

[0037] (1) Acidify graphene with concentrated nitric acid, disperse 400mg graphene in 40mL concentrated nitric acid, react at 60°C for 7 hours to obtain acidified graphene, and then fully wash acidified graphene with deionized water until the washing liquid is Until neutral, vacuum dry at 50°C;

[0038] (2) Add 150 mg of the acidified graphene obtained in step (1) to 40 mL of ethylene glycol, add 405 mg of ferric chloride hexahydrate, ultrasonically disperse for 1 hour, then add 0.15 g of trisodium citrate, 1.8 g of sodium acetate and 1.0 g of Polyethylene glycol, stirred at room temperature for 0.5 hours, then transferred the mixture to a polytetrafluoroethylene-lined stainless steel reactor, and reacted at 200°C for 12 hours to obtain magnetic graphene, and the produc...

Embodiment 2

[0047] Embodiment 2: The magnetic graphene surface that obtains in embodiment 1 coats poly(diallyl dimethyl ammonium chloride) and nano-gold particles and the hydrophilic composite nanomaterial that modifies glucose is applied to low-concentration horseradish Enrichment of oxidase HRP hydrolyzate and detection by MALDI-TOF MS

[0048] (1) Prepare standard proteolysis solution: Accurately weigh 1 mg HRP standard protein, prepare a standard protein solution with a concentration of 5 mg / mL with 25 mM ammonium bicarbonate solution, and boil for ten minutes; add dithiothreitol to make the two The final concentration of thiothreitol was 5 mM, reacted at 60°C for 1 hour, then added iodoacetamide to make the final concentration of iodoacetamide 12.5mM, and reacted for 1 hour at 37°C in the dark; then diluted with 25mM ammonium bicarbonate solution to make the final HRP The concentration is 1mg / mL, according to the ratio of trypsin and standard protein with a mass ratio of 1:40, add tr...

Embodiment 3

[0054] Embodiment 3: The magnetic graphene surface that obtains in embodiment 1 coats poly(diallyl dimethyl ammonium chloride) and nano-gold particles and the hydrophilic composite nanomaterial that modifies glucose as solid-phase microextraction adsorption separation The medium is used for the enrichment and MALDI-TOF MS detection of the mixed solution of HRP enzymolysis solution and bovine serum albumin (BSA) enzymolysis solution

[0055] (1) Prepare standard proteolysis solution: Accurately weigh 1 mg HRP standard protein, prepare a standard protein solution with a concentration of 5 mg / mL with 25 mM ammonium bicarbonate solution, and boil for ten minutes; add dithiothreitol to make the two The final concentration of thiothreitol was 5 mM, reacted at 60°C for 1 hour, then added iodoacetamide to make the final concentration of iodoacetamide 12.5mM, and reacted for 1 hour at 37°C in the dark; then diluted with 25mM ammonium bicarbonate solution to make the final HRP The conce...

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