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Mass Spectrometry-based Protein Site-Specific Glycosylation Analysis 
​Despite significant recent advances in mass spectrometry (MS)-based protein site-specific glycosylation analysis and glycoproteomics, confident identification of glycopeptides is still far from routine. Current practice utilizes high end MS instrument to acquire as many MS2 throughout an LC-MS/MS run and then subject the processed glycopeptide MS2 dataset to database search, followed by scoring and assigning the best matched glycan composition and peptide backbone combination. 
  • The commercially available computational tool, Byonic, is among the most commonly used software that is applicable to both N- and O-glycopeptides. It can search for N- and O-glycopeptides separately or simultaneously. For N-glycopeptides, Y1 ion is considered and scored but not used as a pre-requisite to fix the peptide backbone, thus allowing variables on both peptide and glycan moieties. Overall, it is powerful and reliable when stringent cut-off criteria is applied, but typcially contains many mis-assignments particularly on the glycan part. 
  • The newly introduced pGlyco3 is one of those that offers great potentials to complement Byonic by taking a different glycan-first search strategy. It reflects a growing need to better address the accuracy of glycan assignment instead of reporting as many total hits. 
  • We have set out to investigate how pGlyco3 can be best utilized to corroborate or otherwise refute the positive spectrum matches from search results of Byonic. For O-glycopeptides, we have additionally evaluated O-pair search, which was specifically developed for handling multiply O-glycosylated peptides from mucin, with great emphasis on site localization.
  • Features investigated include those contributing to unique positive matches by one but not the other software, discrepancies in assigned glycan/peptide combination for the same MS2 scan, merging of HCD and EThcD spectra derived from the same glycopeptide precursors prior to searching versus post-search/identification pairing, efficiency in handling multiply N- and/or O-glycosylated peptide, and site localization.
  • For O-glycopeptides, O-pair 
  • Above all, data analysis workflow was developed to seamlessly incorporate results output from both software for cross validation. 
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  • Home
  • Research
    • Glycomics >
      • Glycomics Workflow
      • Mouse Brain
      • Zebrafish
      • Publications
    • Glycoproteomics >
      • Workflow Development
      • Publications
  • People
    • Khoo KH
    • Lab
    • Alumni
  • Resources
    • GlycoMeeting Calendar
    • Essentials of Glycobiology
    • Symbol Nomenclature
    • GlycosylT Handbook
    • Useful Links
    • Videos