Journal
JOURNAL OF PROTEOME RESEARCH
Volume 13, Issue 12, Pages 5561-5569Publisher
AMER CHEMICAL SOC
DOI: 10.1021/pr5005482
Keywords
mass spectrometry; exoglycosidase treatment; microheterogeneity; N-glycans; permethylation; MSn structural analysis glycopeptides; hepatocellular carcinoma; fucosylation; site specificity
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Funding
- NCI [U01 CA168926, RO1 CA135069]
- CCSG [P30 CA51008]
- AMVIS Czech Republic-U.S. exchange program [LH13051]
- Grant Agency of the Czech Republic [P206/12/0503]
- Charles University [ProjectUNCE_204025/2012]
- UNH Glycomics Center
- Glycan Connections, LLC
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Chronic liver diseases are a serious health problem worldwide. One of the frequently reported glycan alterations in liver disease is aberrant fucosylation, which was suggested as a marker for noninvasive serologic monitoring. We present a case study that compares site specific glycoforms of four proteins including haptoglobin, complement factor H, kininogen-1, and hemopexin isolated from the same patient. Our exoglycosidase-assisted LC-MS/MS analysis confirms the high degree of fucosylation of some of the proteins but shows that microheterogeneity is protein- and site-specific. MSn analysis of permethylated detached glycans confirms the presence of LeY glycoforms on haptoglobin, which cannot be detected in hemopexin or complement factor H; all three proteins carry Lewis and H epitopes. Core fucosylation is detectable in only trace amounts in haptoglobin but with confidence on hemopexin and complement factor H, where core fucosylation of the bi-antennary glycans on select glycopeptides reaches 15-20% intensity. These protein-specific differences in fucosylation, observed in proteins isolated from the same patient source, suggest that factors other than up-regulation of enzymatic activity regulate the microheterogeneity of glycoforms. This has implications for selection of candidate proteins for disease monitoring and suggests that site-specific glycoforms have structural determinants, which could lead to functional consequences for specific subsets of proteins or their domains.
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