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dc.contributor.authorParker, Benjamin L.
dc.contributor.authorThaysen-Andersen, Morten
dc.contributor.authorSolis, Nestor
dc.contributor.authorScott, Nichollas E.
dc.contributor.authorLarsen, Martin R.
dc.contributor.authorGraham, Mark E.
dc.contributor.authorPacker, Nicolle H.
dc.contributor.authorCordwell, Stuart J.
dc.date.accessioned2017-05-11T04:58:20Z
dc.date.available2017-05-11T04:58:20Z
dc.date.issued2013
dc.identifier.issn1535-3893
dc.identifier.doi10.1021/pr400783j
dc.identifier.urihttp://hdl.handle.net/10072/336550
dc.description.abstractA combined glycomics and glycoproteomics strategy was developed for the site-specific analysis of N-linked glycosylation heterogeneity from a complex mammalian protein mixture. Initially, global characterization of the N-glycome was performed using porous graphitized carbon liquid chromatography–tandem mass spectrometry (PGC-LC–MS/MS) and the data used to create an N-glycan modification database. In the next step, tryptic glycopeptides were enriched using zwitterionic hydrophilic interaction liquid chromatography (Zic-HILIC) and fractionated by reversed-phase liquid chromatography (RPLC; pH 7.9). The resulting fractions were each separated into two equal aliquots. The first set of aliquots were treated with peptide-N-glycosidase F (PNGase F) to remove N-glycans and the former N-glycopeptides analyzed by nano-RPLC-MS/MS (pH 2.7) and identified by Mascot database search. This enabled the creation of a glycopeptide-centric concatenated database for each fraction. The second set of aliquots was analyzed directly by nanoRPLC-MS/MS (pH 2.7), employing fragmentation by CID and HCD. The assignment of glycan compositions to peptide sequences was achieved by searching the N-glycopeptide HCD MS/MS spectra against the glycopeptide-centric concatenated databases employing the N-glycan modification database. CID spectra were used to assign glycan structures identified in the glycomic analysis to peptide sequences. This multidimensional approach allowed confident identification of 863 unique intact N-linked glycopeptides from 161 rat brain glycoproteins.
dc.description.peerreviewedYes
dc.languageEnglish
dc.language.isoeng
dc.publisherAmerican Chemical Society
dc.relation.ispartofpagefrom5791
dc.relation.ispartofpageto5800
dc.relation.ispartofissue12
dc.relation.ispartofjournalJournal of Proteome Research
dc.relation.ispartofvolume12
dc.subject.fieldofresearchBiochemistry and Cell Biology not elsewhere classified
dc.subject.fieldofresearchChemical Sciences
dc.subject.fieldofresearchBiological Sciences
dc.subject.fieldofresearchcode060199
dc.subject.fieldofresearchcode03
dc.subject.fieldofresearchcode06
dc.titleSite-specific glycan-peptide analysis for determination of N-glycoproteome heterogeneity
dc.typeJournal article
dc.type.descriptionC1 - Articles
dc.type.codeC - Journal Articles
gro.hasfulltextNo Full Text
gro.griffith.authorPacker, Nicki


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