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dc.contributor.authorJensen, Pia H
dc.contributor.authorKarlsson, Niclas G
dc.contributor.authorKolarich, Daniel
dc.contributor.authorPacker, Nicolle H
dc.date.accessioned2017-05-11T05:46:56Z
dc.date.available2017-05-11T05:46:56Z
dc.date.issued2012
dc.identifier.issn1754-2189
dc.identifier.doi10.1038/nprot.2012.063
dc.identifier.urihttp://hdl.handle.net/10072/336567
dc.description.abstractThis protocol shows how to obtain a detailed glycan compositional and structural profile from purified glycoproteins or protein mixtures, and it can be used to distinguish different isobaric glycan isomers. Glycoproteins are immobilized on PVDF membranes before the N-glycans are enzymatically released by PNGase F, isolated and reduced. Subsequently, O-glycans are chemically released from the same protein spot by reductive β-elimination. After desalting with cation exchange microcolumns, the glycans are separated and analyzed by porous graphitized carbon liquid chromatography–electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). Optionally, the glycans can be treated with sialidases or other specific exoglycosidases to yield more detailed structural information. The sample preparation takes approximately 4 d, with a heavier workload on days 2 and 3, and a lighter load on days 1 and 4. The time for data interpretation depends on the complexity of the samples analyzed. This method can be used in conjunction with the analysis of enriched glycopeptides by capillary/nanoLC-ESI-MS/MS, which together provide detailed information regarding the site heterogeneity of glycosylation.
dc.description.peerreviewedYes
dc.languageEnglish
dc.publisherNature Publishing Group
dc.relation.ispartofpagefrom1299
dc.relation.ispartofpageto1310
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.titleStructural analysis of N- and O-glycans released from 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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