Determination of site-specific glycan heterogeneity on glycoproteins

Nat Protoc. 2012 Jun 7;7(7):1285-98. doi: 10.1038/nprot.2012.062.


The comprehensive analysis of protein glycosylation is a major requirement for understanding glycoprotein function in biological systems, and is a prerequisite for producing recombinant glycoprotein therapeutics. This protocol describes workflows for the characterization of glycopeptides and their site-specific heterogeneity, showing examples of the analysis of recombinant human erythropoietin (rHuEPO), α1-proteinase inhibitor (A1PI) and immunoglobulin (IgG). Glycoproteins of interest can be proteolytically digested either in solution or in-gel after electrophoretic separation, and the (glyco)peptides are analyzed by capillary/nano-liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). If required, specific glycopeptide enrichment steps, such as hydrophilic interaction liquid chromatography (HILIC), can also be performed. Particular emphasis is placed on data interpretation and the determination of site-specific glycan heterogeneity. The described workflow takes approximately 3-5 d, including sample preparation and data analysis. The data obtained from analyzing released glycans of rHuEPO and IgG, described in the second protocol of this series (10.1038/nprot.2012.063), provide complementary detailed glycan structural information that facilitates characterization of the glycopeptides.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Chromatography, Liquid / methods
  • Electrophoresis, Polyacrylamide Gel
  • Erythropoietin / chemistry
  • Erythropoietin / metabolism
  • Glycoproteins / chemistry*
  • Glycoproteins / metabolism
  • Glycosylation
  • Humans
  • Polysaccharides / chemistry*
  • Polysaccharides / metabolism
  • Proteomics / methods*
  • Software
  • Tandem Mass Spectrometry / methods


  • EPO protein, human
  • Glycoproteins
  • Polysaccharides
  • Erythropoietin