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dc.contributor.authorKolarich, Daniel
dc.contributor.authorJensen, Pia H
dc.contributor.authorAltmann, Friedrich
dc.contributor.authorPacker, Nicolle H
dc.date.accessioned2017-05-11T05:45:21Z
dc.date.available2017-05-11T05:45:21Z
dc.date.issued2012
dc.identifier.issn1754-2189
dc.identifier.doi10.1038/nprot.2012.062
dc.identifier.urihttp://hdl.handle.net/10072/336566
dc.description.abstractThe 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.
dc.description.peerreviewedYes
dc.languageEnglish
dc.publisherNature Publishing Group
dc.relation.ispartofpagefrom1285
dc.relation.ispartofpageto1298
dc.relation.ispartofissue7
dc.relation.ispartofjournalNature Protocols
dc.relation.ispartofvolume7
dc.subject.fieldofresearchBiochemistry and Cell Biology not elsewhere classified
dc.subject.fieldofresearchBiological Sciences
dc.subject.fieldofresearchMedical and Health Sciences
dc.subject.fieldofresearchChemical Sciences
dc.subject.fieldofresearchcode060199
dc.subject.fieldofresearchcode06
dc.subject.fieldofresearchcode11
dc.subject.fieldofresearchcode03
dc.titleDetermination of site-specific glycan heterogeneity on glycoproteins
dc.typeJournal article
dc.type.descriptionC1 - Articles
dc.type.codeC - Journal Articles
gro.hasfulltextNo Full Text
gro.griffith.authorKolarich, Daniel
gro.griffith.authorPacker, Nicki


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