High-Throughput Analyses of Glycans, Glycosites, and Intact Glycopeptides Using C4-and C18/MAX-Tips and Liquid Handling System

Curr Protoc. 2021 Jul;1(7):e186. doi: 10.1002/cpz1.186.

Abstract

Protein glycosylation is one of the most common and diverse modifications. Aberrant protein glycosylation has been reported to associate with various diseases. High-throughput and comprehensive characterization of glycoproteins is crucial for structural and functional studies of altered glycosylation in biological, physiological, and pathological processes. In this protocol, we detail a workflow for comprehensive analyses of intact glycopeptides (IGPs), glycosylation sites, and glycans from N-linked glycoproteins. By utilizing liquid handling systems, our workflow could enrich IGPs in a high-throughput manner while reducing sample processing time and human error involved in traditional proteomics sample processing techniques. Together, our workflow enables a high-throughput enrichment of glycans, glycosites, and intact glycopeptides from complex biological or clinical samples. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Enzymatic digestion of glycoproteins using C4-tips Basic Protocol 2: Intact glycopeptide analysis using C18/MAX-tips Basic Protocol 3: Glycan and glycosite analysis.

Keywords: glycoproteomics; high-throughput; mass spectrometry; sample processing.

MeSH terms

  • Glycopeptides* / metabolism
  • Glycoproteins
  • Glycosylation
  • Humans
  • Polysaccharides
  • Tandem Mass Spectrometry*

Substances

  • Glycopeptides
  • Glycoproteins
  • Polysaccharides