PXD004243

PXD004243 is an original dataset announced via ProteomeXchange.

Title	Automated N-glycopeptide identification in glycoproteomics
Description	Recent advances in software-driven glycopeptide identification in LC-MS/MS-based N-glycoproteomics have facilitated biochemical studies reporting thousands of intact N-glycopeptides, i.e. N-glycan-conjugated peptides, but the automated identification process remains to be scrutinized. Herein, we explore the efficiency of site-specific glycoprofiling using the PTM-centric search-engine Byonic relative to manual expert annotation. To allow an appropriately deep comparison, the study utilised typical glycoproteomics acquisition and data analysis strategies, but of a single glycoprotein, the uncharacterised N-glycosylated (Asn160, Asn268 and Asn302) 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 acquisition of tryptic N-glycopeptides and released N-glycans. The basigin N-glycosylation sites, which showed extensive micro- and macro-heterogeneity, were then glycoprofiled using Byonic with or without a background of complex peptides using Q-Exactive Orbitrap HCD-MS/MS data. The glycoprofiling efficiencies were assessed against the site-specific reference glycoprofiles and target and decoy proteome databases. The search criteria and confidence thresholds (Byonic scores) recommended by the vendor provided very 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 glycoprofiling efficiency and analysis time. Automated identification of peptide modifications (methionine oxidation/carbamidomethylation) that coincide with monosaccharide mass differences (Fuc/Hex/HexNAc) and accurately distinguishing isobaric (Hex1NeuAc1-R/Fuc1NeuGc1-R) or near-isobaric (NeuAc1-R/Fuc2-R) monosaccharide sub-compositions remain challenging, arguing particular attention to such “difficult-to-identify” N-glycopeptides. The presented analysis provides valuable insights into automated glycopeptide identification; knowledge that facilitates further developments in FDR-based glycoproteomics.
HostingRepository	PRIDE
AnnounceDate	2024-10-22
AnnouncementXML	Submission_2024-10-22_04:22:38.025.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Ling Lee
SpeciesList	scientific name: Homo sapiens (Human); NCBI TaxID: 9606;
ModificationList	monohydroxylated residue; complex glycosylation; iodoacetamide derivatized residue; deamidated residue
Instrument	Bruker Daltonics HCT Series; Q Exactive

Revision	Datetime	Status	ChangeLog Entry
0	2016-05-31 01:46:38	ID requested
1	2016-08-17 00:33:16	announced
⏵ 2	2024-10-22 04:22:40	announced	2024-10-22: Updated project metadata.

Lee LY, Moh ES, Parker BL, Bern M, Packer NH, Thaysen-Andersen M, Toward Automated N-Glycopeptide Identification in Glycoproteomics. J Proteome Res, 15(10):3904-3915(2016) [pubmed]

curator keyword: Technical

submitter keyword: basigin, Byonic, glycoproteomics, glycopeptide,N-glycosylation, LC-MS/MS

Morten Andersen
contact affiliation	Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, Australia
contact email	morten.andersen@mq.edu.au
lab head
Ling Lee
contact affiliation	Macquarie University
contact email	lingyenlee@gmail.com
dataset submitter

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PRIDE project URI

• PRIDE
  1. PXD004243
     1. Label: PRIDE project
     2. Name: Automated N-glycopeptide identification in glycoproteomics