Toward Automated N-Glycopeptide Identification in Glycoproteomics

Ling Y. Lee, Edward S X Moh, Benjamin L. Parker, Marshall Bern, Nicolle H. Packer, Morten Thaysen-Andersen*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

55 Citations (Scopus)


Advances in software-driven glycopeptide identification have facilitated N-glycoproteomics studies reporting thousands of intact N-glycopeptides, i.e., N-glycan-conjugated peptides, but the automated identification process remains to be scrutinized. Herein, we compare the site-specific glycoprofiling efficiency of the PTM-centric search engine Byonic relative to manual expert annotation utilizing typical glycoproteomics acquisition and data analysis strategies but with a single glycoprotein, the uncharacterized multiple N-glycosylated human basigin. Detailed site-specific reference glycoprofiles of purified basigin were manually established using ion-trap CID-MS/MS and high-resolution Q-Exactive Orbitrap HCD-MS/MS of tryptic N-glycopeptides and released N-glycans. The micro- and macroheterogeneous basigin N-glycosylation was site-specifically glycoprofiled using Byonic with or without a background of complex peptides using Q-Exactive Orbitrap HCD-MS/MS. The automated glycoprofiling efficiencies were assessed against the site-specific reference glycoprofiles and target/decoy proteome databases. Within the limits of this single glycoprotein analysis, the search criteria and confidence thresholds (Byonic scores) recommended by the vendor provided high glycoprofiling accuracy and coverage (both >80%) and low peptide FDRs (<1%). The data complexity, search parameters including search space (proteome/glycome size), mass tolerance and peptide modifications, and confidence thresholds affected the automated glycoprofiling efficiency and analysis time. Correct identification of ambiguous peptide modifications (methionine oxidation/carbamidomethylation) whose mass differences coincide with several monosaccharide mass differences (Fuc/Hex/HexNAc) and of ambiguous isobaric (Hex1NeuAc1-R/Fuc1NeuGc1-R) or near-isobaric (NeuAc1-R/Fuc2-R) monosaccharide subcompositions remains challenging in automated glycoprofiling, arguing particular attention paid to N-glycopeptides displaying such "difficult-to-identify" features. This study provides valuable insights into the automated glycopeptide identification process, stimulating further developments in FDR-based glycoproteomics.

Original languageEnglish
Pages (from-to)3904-3915
Number of pages12
JournalJournal of Proteome Research
Issue number10
Publication statusPublished - 7 Oct 2016


  • N-glycosylation
  • LC-MS/MS
  • glycopeptide
  • glycoproteomics
  • basigin
  • glycoprofiling
  • glycomics
  • automated glycopeptide identification
  • Byonic

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