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Concurrent Automated Sequencing of the Glycan and Peptide Portions of O‑Linked Glycopeptide Anions by Ultraviolet Photodissociation Mass Spectrometry
journal contribution
posted on 2016-02-18, 19:07 authored by James A. Madsen, Byoung
Joon Ko, Hua Xu, Jeremy A. Iwashkiw, Scott A. Robotham, Jared B. Shaw, Mario F. Feldman, Jennifer S. BrodbeltO-Glycopeptides are often acidic owing to the frequent occurrence
of acidic saccharides in the glycan, rendering traditional proteomic
workflows that rely on positive mode tandem mass spectrometry (MS/MS)
less effective. In this report, we demonstrate the utility of negative
mode ultraviolet photodissociation (UVPD) MS for the characterization
of acidic O-linked glycopeptide anions. This method
was evaluated for a series of singly and multiply deprotonated glycopeptides
from the model glycoprotein kappa casein, resulting in production
of both peptide and glycan product ions that afforded 100% sequence
coverage of the peptide and glycan moieties from a single MS/MS event.
The most abundant and frequent peptide sequence ions were a/x-type products which, importantly, were
found to retain the labile glycan modifications. The glycan-specific
ions mainly arose from glycosidic bond cleavages (B, Y, C, and Z ions)
in addition to some less common cross-ring cleavages. On the basis
of the UVPD fragmentation patterns, an automated database searching
strategy (based on the MassMatrix algorithm) was designed that is
specific for the analysis of glycopeptide anions by UVPD. This algorithm
was used to identify glycopeptides from mixtures of glycosylated and
nonglycosylated peptides, sequence both glycan and peptide moieties
simultaneously, and pinpoint the correct site(s) of glycosylation.
This methodology was applied to uncover novel site-specificity of
the O-linked glycosylated OmpA/MotB from the “superbug” A. baumannii to help aid in the elucidation of the functional
role that protein glycosylation plays in pathogenesis.
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model glycoprotein kappa caseinproteomic workflowsglycan moietiesConcurrent Automated Sequencingglycan modificationsnonglycosylated peptidesmode tandem mass spectrometryPeptide PortionsUVPD fragmentation patternsglycosidic bond cleavagesMSprotein glycosylationZ ionspeptide moietiesdeprotonated glycopeptidesMassMatrix algorithmacidic saccharidesglycan product ionspeptide sequence ionsglycopeptide anions
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