Mapping site-specific protein N-glycosylations through liquid chromatography/mass spectrometry and targeted tandem mass spectrometry

Rapid Commun Mass Spectrom. 2010 Apr 15;24(7):965-72. doi: 10.1002/rcm.4474.


Glycosylation is one of the most common posttranslational modifications (PTMs) of proteins, the characterization of which is commonly achieved through proteomic protocol, involving trypsin digestion followed by liquid chromatography/tandem mass spectrometry (LC/MS/MS). However, it is often not possible to characterize all glycopeptides in a complex sample because of the high complexity of glycoproteomic samples, and the relative lower abundances of glycopeptides in comparison to the unmodified peptides. We present here a targeted MS/MS analysis approach, which utilizes a previously developed computational tool, GlyPID, to guide multiple experiments, thus permitting a complete characterization of all N-glycosylation sites of glycoproteins present in a complex sample. We have tested our approach using model glycoproteins analyzed by high-resolution LTQ-FT MS. The results demonstrate a potential use of our method for a high-throughput characterization of complex mixtures of glycosylated proteins.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Algorithms
  • Animals
  • Cattle
  • Chromatography, Liquid / methods*
  • Complex Mixtures / chemistry
  • Computational Biology / methods*
  • Glycopeptides / chemistry*
  • Glycopeptides / metabolism
  • Glycosylation
  • Humans
  • Peptide Mapping / methods*
  • Protein Processing, Post-Translational
  • Reproducibility of Results
  • Software*
  • Tandem Mass Spectrometry / methods*
  • alpha-Fetoproteins / chemistry


  • Complex Mixtures
  • Glycopeptides
  • alpha-Fetoproteins