Title : Determination of site-specific glycan heterogeneity on glycoproteins
Abstract :
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