Site-specific glycan-peptide analysis for determination of N-glycoproteome heterogeneity

J Proteome Res. 2013 Dec 6;12(12):5791-800. doi: 10.1021/pr400783j. Epub 2013 Nov 1.


A combined glycomics and glycoproteomics strategy was developed for the site-specific analysis of N-linked glycosylation heterogeneity from a complex mammalian protein mixture. Initially, global characterization of the N-glycome was performed using porous graphitized carbon liquid chromatography-tandem mass spectrometry (PGC-LC-MS/MS) and the data used to create an N-glycan modification database. In the next step, tryptic glycopeptides were enriched using zwitterionic hydrophilic interaction liquid chromatography (Zic-HILIC) and fractionated by reversed-phase liquid chromatography (RPLC; pH 7.9). The resulting fractions were each separated into two equal aliquots. The first set of aliquots were treated with peptide-N-glycosidase F (PNGase F) to remove N-glycans and the former N-glycopeptides analyzed by nano-RPLC-MS/MS (pH 2.7) and identified by Mascot database search. This enabled the creation of a glycopeptide-centric concatenated database for each fraction. The second set of aliquots was analyzed directly by nanoRPLC-MS/MS (pH 2.7), employing fragmentation by CID and HCD. The assignment of glycan compositions to peptide sequences was achieved by searching the N-glycopeptide HCD MS/MS spectra against the glycopeptide-centric concatenated databases employing the N-glycan modification database. CID spectra were used to assign glycan structures identified in the glycomic analysis to peptide sequences. This multidimensional approach allowed confident identification of 863 unique intact N-linked glycopeptides from 161 rat brain glycoproteins.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Brain / metabolism*
  • Brain Chemistry
  • Carbohydrate Sequence
  • Chromatography, Liquid / instrumentation
  • Chromatography, Liquid / methods
  • Databases, Factual
  • Genetic Heterogeneity
  • Glycomics / instrumentation
  • Glycomics / methods*
  • Glycosylation
  • Humans
  • Hydrogen-Ion Concentration
  • Male
  • Molecular Sequence Annotation
  • Molecular Sequence Data
  • Peptide Mapping / methods*
  • Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / chemistry
  • Protein Processing, Post-Translational*
  • Proteome / analysis*
  • Proteome / chemistry
  • Rats
  • Rats, Inbred Lew
  • Tandem Mass Spectrometry


  • Proteome
  • Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase