[Sample pre-processing for mass spectrometric analysis of glycoprotein structure].
A novel, simple and fast method based on a quadrupole ion trap (QIT) and a time-of-flight mass spectrograph was developed for the glycosylation site and glycan structure analysis of glycoprotein by in-solution digestion and in-gel digestion which did not need to enrich and label the glycopeptide. Horseradish peroxidase (HRP) and RNase B were utilised to optimize the conditions of sample pre-processing. The effects of the conditions were investigated separately and the optimal conditions were: heat denaturation of RNase B, chemical denaturation of HRP, RNase B digestion with endoproteinase Lys-C, HRP digestion with trypsin, 12 - 16 h of digestion, 50% acetonitrile-5% trifluoroacetic acid of the extraction solution, sandwich spotting method of the sample.
Zhou
Wei
W
Institutes of Biomedical Sciences, Fudan University, Shanghai 200032, China. echozhou926@gmail.com
Liu
Xiaohui
X
Zhou
Xinwen
X
Shen
Huali
H
Yang
Pengyuan
P
D006023:Glycoproteins / Q000737:chemistry* / Q000378:metabolism*; D006735:Horseradish Peroxidase / Q000378:metabolism; D013058:Mass Spectrometry / Q000379:methods*; D008666:Metalloendopeptidases / Q000378:metabolism; D012260:Ribonucleases / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D014357:Trypsin / Q000378:metabolism
D006023:Glycoproteins; D006735:Horseradish Peroxidase; D012260:Ribonucleases; C029600:ribonuclease B; D014357:Trypsin; D008666:Metalloendopeptidases; C039830:peptidyl-Lys metalloendopeptidase