Glycan side reaction may compromise ETD-based glycopeptide identification

J Am Soc Mass Spectrom. 2014 Jun;25(6):977-87. doi: 10.1007/s13361-014-0852-9. Epub 2014 Mar 25.

Abstract

Tris(hydroxymethyl)aminomethane (Tris) is one of the most frequently used buffer ingredients. Among other things, it is recommended and is usually used for lectin-based affinity enrichment of glycopeptides. Here we report that sialic acid, a common 'capping' unit in both N- and O-linked glycans may react with this chemical, and this side reaction may compromise glycopeptide identification when ETD spectra are the only MS/MS data used in the database search. We show that the modification may alter N- as well as O-linked glycans, the Tris-derivative is still prone to fragmentation both in 'beam-type' CID (HCD) and ETD experiments, at the same time--since the acidic carboxyl group was 'neutralized'--it will display a different retention time than its unmodified counterpart. We also suggest solutions that--when incorporated into existing search engines--may significantly improve the reliability of glycopeptide assignments.

Publication types

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

MeSH terms

  • Animals
  • Chromatography, Affinity
  • Glycopeptides / analysis
  • Glycopeptides / chemistry*
  • Glycopeptides / metabolism
  • Ions / chemistry
  • Ions / metabolism
  • Mice
  • N-Acetylneuraminic Acid / chemistry
  • N-Acetylneuraminic Acid / metabolism
  • Peptide Fragments / analysis
  • Peptide Fragments / chemistry
  • Peptide Fragments / metabolism
  • Proteomics / methods
  • Proteomics / standards*
  • Reproducibility of Results
  • Synaptosomes / chemistry
  • Tandem Mass Spectrometry / methods
  • Tandem Mass Spectrometry / standards*

Substances

  • Glycopeptides
  • Ions
  • Peptide Fragments
  • N-Acetylneuraminic Acid