Journal
NATURE PROTOCOLS
Volume 7, Issue 7, Pages 1285-1298Publisher
NATURE PUBLISHING GROUP
DOI: 10.1038/nprot.2012.062
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Funding
- Erwin Schrodinger Fellowship from the Austrian Science Fund [J2661]
- Macquarie University
- Danish Agency for Science, Technology and Innovation [272-07-0066]
- Austrian Science Fund (FWF) [J2661] Funding Source: Austrian Science Fund (FWF)
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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), alpha 1-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 (LCLC-ESESI-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.
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