One-step enrichment-derivatization treatment and maldi-tof-ms analysis method of glycoprotein n-sugar chains based on graphene
A MALDI-TOF-MS, glycoprotein technology, applied in analytical materials, material separation, measurement devices, etc., can solve the problems of sample loss, complicated operation steps, etc., to improve the signal intensity, improve the mass spectral signal response, small space position blocking effect
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[0045] a) Preparation of graphene oxide. Usually, 0.5g phosphorus flake graphite and 0.5gNa 2 NO 3 Added to 23mL pre-cooled concentrated sulfuric acid. Subsequently, 4gKMnO was slowly added to it 4 . The resulting mixture was stirred in an ice bath for 10 min and then transferred to a 35 °C water bath for stirring. After 1 h, 40 mL of deionized water was slowly added dropwise to the above system while maintaining the temperature of the system at 90 to 95°C. After 1 h, 100 mL of deionized water and 3 mL of H 2 o 2 And the reaction solution was stirred at room temperature for 2 h. Subsequently, the resulting product was washed with 1000 mL of deionized water containing 5 mL of HCl and then with deionized water until the pH of the eluent became neutral. Finally, the obtained product was vacuum-dried at 50° C. for 24 h to obtain the desired graphene oxide.
[0046] b) the above-mentioned graphene oxide obtained with NaBH 4 Carry out reduction treatment to obtain graphene...
Embodiment 1
[0047] Example 1: One-step enrichment-derivatization treatment and MALDI-TOF-MS analysis of standard oligosaccharides based on graphene
[0048] One-step enrichment and derivatization of the standard oligosaccharide maltoheptaose (DP7) based on graphene and PBH.
[0049] 10 pmol of DP7 standard, 500 pmol of PBH (PBH) and 0.1 mg of graphene were mixed in 0.5 mL of methanol containing 0.5% acetic acid, and incubated at 90° C. for 1 h. After discarding the supernatant, the obtained graphene sample was washed three times with deionized water and methanol, respectively, and the enriched sugar chains were eluted with DMF, and analyzed by MALDI-TOF-MS.
[0050] The mass spectrometry conditions are: the instrument is 4800 ProteomicAnalyzer, the positive ion reflection mode is selected (positive reflector mode), the acceleration voltage is 20KV, the scanning range is m / z1000-4000, the laser energy is 6000, and each mass spectrum is composed of a total of 1000 laser emission pulses. Gr...
Embodiment 2
[0054] Example 2: One-step enrichment-derivatization treatment and MALDI-TOF-MS analysis of standard glycoprotein N-glycan chains based on graphene
[0055]Asialo-fetuin was dissolved in 50 mM ammonium bicarbonate (pH=8.0) or phosphate buffer (pH=7.8) at a concentration of 1 mg / mL. Afterwards, heat denaturation at 95°C for 10min, and after cooling, add the required enzyme at the ratio of 1U peptide N-glycosidase F (PNGaseF): 100μg standard glycoprotein (asialofetuin), and bathe in 37°C for 16h to make it Sugar chains are fully released. Then add an appropriate amount of PBH and graphene (wherein, the ratio of graphene and PBH is 1mg: 15nmol; the molar ratio of PBH and sugar chains is 50: 1) in the system, and the resulting mixture is contained in a mass concentration of 0.5% Incubate in acetic acid in methanol for 1 h at 90°C. After discarding the supernatant, the obtained graphene sample precipitate was washed three times with deionized water and methanol respectively, and ...
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