An improved protocol for N-glycosylation analysis of gel-separated sialylated glycoproteins by MALDI-TOF/TOF.
Different glycoforms of some proteins have been identified as differential spots for certain diseases in 2-DE, indicating disease-related glycosylation changes. It is routine to determine the site-specific glycosylation of nonsialylated N-glycoproteins from a single gel spot, but some obstacles still exist in analyzing sialylated glycoproteins due to the lability and higher detection limit of acid glycans in MALDI-TOF/TOF analysis. Thus, we present an improved protocol here. Tryptic glycopeptides were separated and subjected to MALDI-TOF/TOF analysis, resulting in the identification of site-specific glycosylation of high-intensity glycopeptides. Sequential deglycosylation and desialylation were used to improve the identification of glycosylation sites and desialylated glycans. The site-specific glycosylation of large glycopeptides and low-intensity glycopeptides was deduced based on the masses of glycopeptides, deglycosylated peptides and desialylated glycans. By applying it to 2-DE separated human serum, the difference of N-glycosylation was successfully determined for α1-antitrypsin between different gel spots.
Beijing institute of Genomics, Chinese Academy of Science, Beijing, China. hpiliang@163.com
Piliang
P
Hao
Yan
Y
Ren
Yongming
Y
Xie
D001665:Binding Sites; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D006020:Glycopeptides / Q000032:analysis*; D006023:Glycoproteins / Q000032:analysis*; D006031:Glycosylation; D006801:Humans; D011134:Polysaccharides / Q000378:metabolism; D015203:Reproducibility of Results; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D014168:Transferrin / Q000378:metabolism; D000515:alpha 1-Antitrypsin / Q000378:metabolism
D006020:Glycopeptides; D006023:Glycoproteins; D011134:Polysaccharides; D014168:Transferrin; D000515:alpha 1-Antitrypsin