Determination of site-specific glycan heterogeneity on glycoproteins.
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.
Biomolecular Frontiers Research Centre, Faculty of Science, Macquarie University, Sydney, New South Wales, Australia.
Daniel
D
Kolarich
Pia H
PH
Jensen
Friedrich
F
Altmann
Nicolle H
NH
Packer
D002853:Chromatography, Liquid / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D004921:Erythropoietin / Q000737:chemistry / Q000378:metabolism; D006023:Glycoproteins / Q000737:chemistry* / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D011134:Polysaccharides / Q000737:chemistry* / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D012984:Software; D053719:Tandem Mass Spectrometry / Q000379:methods
C554270:EPO protein, human; D006023:Glycoproteins; D011134:Polysaccharides; D004921:Erythropoietin