Glycosylation Profiling of Glycoproteins Secreted from Cultured Cells Using Glycan Node Analysis and GC-MS

Jesús S. Aguilar Díaz de león, Chad R. Borges

Research output: Chapter in Book/Report/Conference proceedingChapter

Abstract

Glycan “node” analysis is the process by which pooled glycans within complex biological samples are chemically deconstructed in a way that facilitates the analytical quantification of uniquely linked monosaccharide units (glycan “nodes”). It is based on glycan methylation analysis (a.k.a. linkage analysis) that has historically been applied to pre-isolated glycans. Thus, when using glycan node analysis, unique glycan features within whole biospecimens such as “core fucosylation,” “α2-6 sialylation,” “β1-6 branching,” “β1-4 branching,” and “bisecting GlcNAc,” are captured as single analytical signals by GC-MS. Here we describe the use of this methodology in cell culture supernatant and in the analysis of IgG (alpha-1 antitrypsin) glycans. The effect of IL-6 and IL-1β cytokines on secreted hepatocyte protein glycan features is demonstrated; likewise, the impact of neuraminidase treatment of IgG is illustrated. For the majority of glycan nodes, the assay is consistent and reproducible on a day-to-day basis; because of this, relatively subtle shifts in the relative abundance of glycan features can be captured using this approach.

Original languageEnglish (US)
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Pages317-330
Number of pages14
DOIs
StatePublished - 2021

Publication series

NameMethods in Molecular Biology
Volume2271
ISSN (Print)1064-3745
ISSN (Electronic)1940-6029

Keywords

  • Aberrant Glycosylation
  • Antibody Glycosylation Profiling
  • Cell Culture Supernatant
  • GC-MS
  • Glycan Nodes
  • Glycan Permethylation
  • Glycans
  • Glycosylation Profiling
  • Secreted Glycoproteins

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

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