Identification of Glycosylated Peptides Using Hydrophilic Nanocomposites Combined with Mass Spectrometry
A nanocomposite material and mass spectrometry analysis technology, applied in the field of enrichment and detection of glycosylated peptides, to achieve high sensitivity, high magnification, and good selectivity
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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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