Development of a combined chemical and enzymatic approach for the mass spectrometric identification and quantification of aberrant N-glycosylation.
Direct mass spectrometric analysis of aberrant protein glycosylation is a challenge to the current analytical techniques. Except lectin affinity chromatography, no other glycosylation enrichment techniques are available for analysis of aberrant glycosylation. In this study, we developed a combined chemical and enzymatic strategy as an alternative for the mass spectrometric analysis of aberrant glycosylation. Sialylated glycopeptides were enriched with reverse glycoblotting, cleaved by endoglycosidase F3 and analyzed by mass spectrometry with both neutral loss triggered MS(3) in collision induced dissociation (CID) and electron transfer dissociation (ETD). Interestingly, a great part of resulted glycopeptides were found with fucose attached to the N-acetylglucosamine (N-GlcNAc), which indicated that the aberrant glycosylation that is carrying both terminal sialylation and core fucosylation was identified. Totally, 69 aberrant N-glycosylation sites were identified in sera samples from hepatocellular carcinoma (HCC) patients. Following the identification, quantification of the level of this aberrant glycosylation was also carried out using stable isotope dimethyl labeling and pooled sera sample from liver cirrhosis and HCC was compared. Six glycosylation sites demonstrated elevated level of aberrancy, which demonstrated that our developed strategy was effective in both qualitative and quantitative studies of aberrant glycosylation.
Key Laboratory of Separation Sciences for Analytical Chemistry, National Chromatographic R&A Center, Dalian Institute of Chemical Physics, The Chinese Academy of Sciences, Dalian 116023, China.
Rui
R
Chen
Fangjun
F
Wang
Yexiong
Y
Tan
Zhen
Z
Sun
Chunxia
C
Song
Mingliang
M
Ye
Hongyang
H
Wang
Hanfa
H
Zou
D006528:Carcinoma, Hepatocellular / Q000097:blood*; D005260:Female; D005643:Fucose / Q000378:metabolism; D006023:Glycoproteins / Q000097:blood*; D006031:Glycosylation; D006801:Humans; D008113:Liver Neoplasms / Q000097:blood*; D008297:Male; D013058:Mass Spectrometry; D019158:N-Acetylneuraminic Acid / Q000378:metabolism; D009363:Neoplasm Proteins / Q000097:blood*
D006023:Glycoproteins; D009363:Neoplasm Proteins; D005643:Fucose; D019158:N-Acetylneuraminic Acid