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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.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.publisherAmerican Chemical Society
dc.relation.ispartofjournalJournal of Proteome Research
dc.subject.fieldofresearchBiochemistry and Cell Biology not elsewhere classified
dc.subject.fieldofresearchChemical Sciences
dc.subject.fieldofresearchBiological Sciences
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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