Large-scale identification of n-linked intact glycopeptides in human serum using HILIC enrichment and spectral library search

Qingbo Shu, Mengjie Li, Lian Shu, Zhiwu An, Jifeng Wang, Hao Lv, Ming Yang, Tanxi Cai, Tony Hu, Yan Fu, Fuquan Yang

Research output: Contribution to journalArticlepeer-review

16 Scopus citations

Abstract

Large-scale identification of N-linked intact glycopeptides by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) in human serum is challenging because of the wide dynamic range of serum protein abundances, the lack of a complete serum N-glycan database and the existence of proteoforms. In this regard, a spectral library search method was presented for the identification of N-linked intact glycopeptides from Nlinked glycoproteins in human serum with target-decoy and motif-specific false discovery rate (FDR) control. Serum proteins were firstly separated into low-abundance and high-abundance proteins by acetonitrile (ACN) precipitation. After digestion, the N-linked intact glycopeptides were enriched by hydrophilic interaction liquid chromatography (HILIC) and a portion of the enriched N-linked intact glycopeptides were processed by Peptide-N-Glycosidase F (PNGase F) to generate N-linked deglycopeptides. Both Nlinked intact glycopeptides and deglycopeptides were analyzed by LC-MS/MS. From N-linked deglycopeptides data sets, 764 N-linked glycoproteins, 1699 N-linked glycosites and 3328 unique N-linked deglycopeptides were identified. Four types of N-linked glycosylation motifs (NXS/T/C/V, X≠P) were used to recognize the N-linked deglycopeptides. The spectra of these N-linked deglycopeptides were utilized for N-linked deglycopeptides library construction and identification of N-linked intact glycopeptides. A database containing 739 N-glycan masses was constructed and utilized during spectral library search for the identification of N-linked intact glycopeptides. In total, 526 N-linked glycoproteins, 1036 N-linked glycosites, 22,677 N-linked intact glycopeptides and 738 N-glycan masses were identified under 1% FDR, representing the most in-depth serum Nglycoproteome identified by LC-MS/MS at N-linked intact glycopeptide level.

Original languageEnglish (US)
Pages (from-to)672-689
Number of pages18
JournalMolecular and Cellular Proteomics
Volume19
Issue number4
DOIs
StatePublished - 2020

ASJC Scopus subject areas

  • Analytical Chemistry
  • Biochemistry
  • Molecular Biology

Fingerprint

Dive into the research topics of 'Large-scale identification of n-linked intact glycopeptides in human serum using HILIC enrichment and spectral library search'. Together they form a unique fingerprint.

Cite this