Glycopeptide Identification with a Comprehensive Proteomics Search Engine - Project Summary/Abstract
Glycoproteomics is the large-scale study of carbohydrate modifications on proteins, including site-specific
glycan heterogeneity. The most commonly used platform for glycoproteomics is liquid chromatography,
electrospray-ionization tandem mass spectrometry (LC-ESI-MS/MS), the same platform used for proteomics,
but glycoproteomics is inherently more difficult than proteomics for a variety of reasons. Glycopeptide signals
are split over many glycoforms, so they tend to be less intense than ordinary peptide signals. Glycopeptides
ionize less easily and produce lower-quality tandem mass spectra, because it is difficult to simultaneously
fragment both the peptide and glycan parts. Finally, glycopeptide data analysis is much more difficult than
ordinary peptide data analysis, due to the explosion of possibilities, for example, 10s to 100s of glycan
compositions per N-linked glycosylation site, and fewer compositions but much more closely spaced sites in
the case of O-linked glycosylation. Despite these challenges, glycoproteomics is becoming increasingly
feasible with improvements in experimental methods (for example, zinc-finger knockouts), data acquisition
modes (such as electron-transfer dissociation), and data analysis software (Byonic search engine). With these
improvements and others, glycoproteomics has the potential to leverage the vast worldwide investment in
proteomics to revolutionize glycobiology.
In this project, we propose to accelerate development of Byonic, the only general-purpose proteomics
search engine with special support for glycopeptide analysis. Specifically, we propose integrated analysis of
complementary fragmentation modes, integrated analysis of MS1 and MS2 (that is, intact and fragmented
peptides), and finally topology (“cartoon”) assignment for glycopeptide spectra.
Glycosylation plays many key biological roles, including cancer markers, immune system recognition, and
sperm/egg binding, so worldwide improvement in glycoproteomics could have great impact for human health.
