Abstract
A MS-based methodology has been developed for analysis of core-fucosylated versus antennary-fucosylated glycosites in glycoproteins. This procedure is applied to the glycoprotein alpha-1-antitrypsin (A1AT), which contains both core- and antennary-fucosylated glycosites. The workflow involves digestion of intact glycoproteins into glycopeptides, followed by double digestion with sialidase and galactosidase. The resulting glycopeptides with truncated glycans were separated using an off-line HILIC (hydrophilic interaction liquid chromatography) separation where multiple fractions were collected at various time intervals. The glycopeptides in each fraction were treated with PNGase F and then divided into halves. One half of the sample was applied for peptide identification while the other half was processed for glycan analysis by derivatizing with a meladrazine reagent followed by MS analysis. This procedure provided site-specific identification of glycosylation sites and the ability to distinguish core fucosylation and antennary fucosylation via a double digestion and a mass profile scan. Both core and antennary fucosylation are shown to be present on various glycosites in A1AT.
Original language | English |
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Pages (from-to) | 2624-2632 |
Number of pages | 9 |
Journal | ELECTROPHORESIS |
Volume | 37 |
Issue number | 20 |
DOIs | |
State | Published - Oct 1 2016 |
Keywords
- Alpha-1-antitrypsin
- Antennary fucosylation
- Core fucosylation
- Glycan structure
- Liquid chromatography-mass spectrometry