In-depth analysis of site-specific N-glycosylation in vitronectin from human plasma by tandem mass spectrometry with immunoprecipitation.
The characterization of site-specific microheterogeneity in glycoprotein is very important for understanding cell biology and disease processes. Vitronectin is well known to be a multifunctional glycoprotein in the blood and the extracellular matrix, which is related to hepatocellular carcinoma (HCC). Here, we systematically analyzed the site-specific N-glycopeptides of vitronectin in human plasma by tandem mass spectrometry combined with immunoprecipitation and hydrophilic interaction liquid chromatography (HILIC) enrichment. Vitronectin was purified with immunoprecipitation by monoclonal antibody from plasma and digested to tryptic N-glycopeptides.Then, enrichment with HILIC materials was used and followed by analysis with nano-LC/MS/MS. The sequences of N-glycopeptides were identified from the mass spectra by high-energy C-trap dissociation (HCD) and collision-induced dissociation (CID). In HCD mode, oxonium ions were used for recognizing glycopeptides and y ions for sequencing the peptide backbone. In CID mode, Y ions were used for characterizing their glycoforms. As a result, a total of 17 site-specific N-glycopeptides were completely identified in all of the three N-glycosylation sites of vitronectin in human plasma, including 12 N-glycopeptides first reported. Finally, we specifically found that three hybrid and four complex glycopeptides of triantennary forms with outer fucosylation increased in HCC human plasma.
Mass Spectrometry Research Center, Korea Basic Science Institute, Chungbuk, Republic of Korea.
Heeyoun
H
Hwang
Ju Yeon
JY
Lee
Hyun Kyoung
HK
Lee
Gun Wook
GW
Park
Hoi Keun
HK
Jeong
Myeong Hee
MH
Moon
Jin Young
JY
Kim
Jong Shin
JS
Yoo
D000595:Amino Acid Sequence; D002240:Carbohydrate Sequence; D006528:Carcinoma, Hepatocellular / Q000097:blood / Q000737:chemistry; D006020:Glycopeptides / Q000032:analysis*; D006031:Glycosylation; D006801:Humans; D047468:Immunoprecipitation / Q000379:methods*; D008113:Liver Neoplasms / Q000097:blood / Q000737:chemistry; D008969:Molecular Sequence Data; D053719:Tandem Mass Spectrometry / Q000379:methods*; D019096:Vitronectin / Q000097:blood* / Q000737:chemistry*
D006020:Glycopeptides; D019096:Vitronectin