Detection and Characterization of Low Abundance Glycopeptides Via Higher-Energy C-Trap Dissociation and Orbitrap Mass Analysis |
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Authors: | Gene Hart-Smith Mark J Raftery |
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Institution: | (1) NSW Systems Biology Initiative, University of New South Wales, Sydney, New South Wales, 2052, Australia;(2) Bioanalytical Mass Spectrometry Facility, University of New South Wales, Sydney, New South Wales, 2052, Australia |
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Abstract: | Broad-scale mass spectrometric analyses of glycopeptides are constrained by the considerable complexity inherent to glycoproteomics,
and techniques are still being actively developed to address the associated analytical difficulties. Here we apply Orbitrap
mass analysis and higher-energy C-trap dissociation (HCD) to facilitate detailed insights into the compositions and heterogeneity
of complex mixtures of low abundance glycopeptides. By generating diagnostic oxonium product ions at mass measurement errors
of <5 ppm, highly selective glycopeptide precursor ion detections are made at sub-fmol limits of detection: analyses of proteolytic
digests of a hen egg glycoprotein mixture detect 88 previously uncharacterized glycopeptides from 666 precursor ions selected
for MS/MS, with only one false positive due to co-fragmentation of a non-glycosylated peptide with a glycopeptide. We also
demonstrate that by (1) identifying multiple series of glycoforms using high mass accuracy single stage MS spectra, and (2)
performing product ion scans at optimized HCD collision energies, the identification of peptide + N-acetylhexosamine (HexNAc) ions (Y1 ions) can be readily achieved at <5 ppm mass measurement errors. These data allow base
peptide sequences and glycan compositional information to be attained with high confidence, even for glycopeptides that produce
weak precursor ion signals and/or low quality MS/MS spectra. The glycopeptides characterized from low fmol abundances using
these methods allow two previously unreported glycosylation sites on the Gallus gallus protein ovoglycoprotein (amino acids 82 and 90) to be confirmed; considerable glycan heterogeneities at amino acid 90 of
ovoglycoprotein, and amino acids 34 and 77 of Gallus gallus ovomucoid are also revealed. |
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