title: Combining high-energy C-trap dissociation and electron transfer dissociation for protein O-GlcNAc modification site assignment.
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.
authors: Complex Carbohydrate Research Center, University of Georgia, Athens, Georgia 30602, United States.
authors: Peng
authors: P
authors: Zhao
authors: Rosa
authors: R
authors: Viner
authors: Chin Fen
authors: CF
authors: Teo
authors: Geert-Jan
authors: GJ
authors: Boons
authors: David
authors: D
authors: Horn
authors: Lance
authors: L
authors: Wells
mesh_terms: D000117:Acetylglucosamine / Q000737:chemistry* / Q000378:metabolism; D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000737:chemistry; D006031:Glycosylation; D057809:HEK293 Cells; D006801:Humans; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry* / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D040901:Proteomics / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D000911:Antibodies, Monoclonal; D010446:Peptide Fragments; D000117:Acetylglucosamine