Novel LC-MS<sup>2</sup> Product Dependent Parallel
Data Acquisition Function and Data Analysis Workflow for Sequencing and Identification of Intact
Glycopeptides
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Abstract
Data dependent acquisition (DDA)
of higher collision energy dissociation
(HCD)-MS<sup>2</sup> followed by electron transfer dissociation (ETD)-MS<sup>2</sup> upon detection of glycan-specific oxonium is one of the better
approaches in current LC-MS<sup>2</sup> analysis of intact glycopeptides.
Although impressive numbers of glycopeptide identification by a direct
database search have been reported, false positives remained high
and difficult to determine. Even in cases when the peptide backbones
were correctly identified, the exact glycan moieties were often erroneously
assigned. Any attempt to fit the best glycosyl composition match by
mass only is problematic particularly when the correct monoisotopic
precursor cannot be determined unambiguously. Taking advantage of
a new trihybrid Orbitrap configuration, we experimented with adding
in a parallel ion trap collision induced dissociation (CID)-MS<sup>2</sup> data acquisition to the original HCD-product dependent (pd)-ETD
function. We demonstrated the feasibility and advantage of identifying
the peptide core ion directly from edited HCD-MS<sup>2</sup> data
as an easy way to reduce false positives without compromising much
sensitivity in intact glycopeptide positive spectrum matches. Importantly,
the additional CID-MS<sup>2</sup> data allows one to validate the
glycan assignment and provides insight into possible glycan modifications.
Moreover, it is a viable alternative to deduce the glycopeptide backbone
particularly in cases when the peptide backbone cannot be identified
by ETD/HCD. The novel HCD-pd-CID/ETD workflow combines the best possible
decision tree dependent MS<sup>2</sup> data acquisition modes currently
available for glycoproteomics within a rapid Top Speed DDA duty cycle.
Additional informatics can conceivably be developed to mine and integrate
the rich information contained within for simultaneous N- and O-glycopeptide
analysis