A procedure for the analysis of site-specific and structure-specific fucosylation in alpha-1-antitrypsin

Haidi Yin, Jianhui Zhu, Jing Wu, Zhijing Tan, Mingrui An, Shiyue Zhou, Yehia Mechref, David M. Lubman

Research output: Contribution to journalArticlepeer-review

7 Scopus citations

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 languageEnglish
Pages (from-to)2624-2632
Number of pages9
JournalELECTROPHORESIS
Volume37
Issue number20
DOIs
StatePublished - Oct 1 2016

Keywords

  • Alpha-1-antitrypsin
  • Antennary fucosylation
  • Core fucosylation
  • Glycan structure
  • Liquid chromatography-mass spectrometry

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