Combining high-energy C-trap dissociation and electron transfer dissociation for protein O-GlcNAc modification site assignment

J Proteome Res. 2011 Sep 2;10(9):4088-104. doi: 10.1021/pr2002726. Epub 2011 Jul 25.

Abstract

Mass spectrometry-based studies of proteins that are post-translationally modified by O-linked β-N-acetylglucosamine (O-GlcNAc) are challenged in effectively identifying the sites of modification while simultaneously sequencing the peptides. Here we tested the hypothesis that a combination of high-energy C-trap dissociation (HCD) and electron transfer dissociation (ETD) could specifically target the O-GlcNAc modified peptides and elucidate the amino acid sequence while preserving the attached GlcNAc residue for accurate site assignment. By taking advantage of the recently characterized O-GlcNAc-specific IgG monoclonal antibodies and the combination of HCD and ETD fragmentation techniques, O-GlcNAc modified proteins were enriched from HEK293T cells and subsequently characterized using the LTQ Orbitrap Velos ETD (Thermo Fisher Scientific) mass spectrometer. In our data set, 83 sites of O-GlcNAc modification are reported with high confidence confirming that the HCD/ETD combined approach is amenable to the detection and site assignment of O-GlcNAc modified peptides. Realizing HCD triggered ETD fragmentation on a linear ion trap/Orbitrap platform for more in-depth analysis and application of this technique to other post-translationally modified proteins are currently underway. Furthermore, this report illustrates that the O-GlcNAc transferase appears to demonstrate promiscuity with regards to the hydroxyl-containing amino acid modified in short stretches of primary sequence of the glycosylated polypeptides.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Acetylglucosamine / chemistry*
  • Acetylglucosamine / metabolism
  • Amino Acid Sequence
  • Antibodies, Monoclonal / chemistry
  • Glycosylation
  • HEK293 Cells
  • Humans
  • Molecular Sequence Data
  • Peptide Fragments / chemistry*
  • Peptide Fragments / metabolism
  • Protein Processing, Post-Translational
  • Proteomics / methods*
  • Tandem Mass Spectrometry / methods*

Substances

  • Antibodies, Monoclonal
  • Peptide Fragments
  • Acetylglucosamine