Combining High-Energy C-Trap Dissociation and Electron Transfer Dissociation for Protein O-GlcNAc Modification Site Assignment
In: Journal of Proteome Research, Jg. 10 (2011-07-25), S. 4088-4104
Online
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Zugriff:
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 dataset, 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.
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Combining High-Energy C-Trap Dissociation and Electron Transfer Dissociation for Protein O-GlcNAc Modification Site Assignment
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Autor/in / Beteiligte Person: | Wells, Lance ; Boons, Geert-Jan ; Viner, Rosa ; Zhao, Peng ; Chin Fen Teo ; Horn, David |
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Zeitschrift: | Journal of Proteome Research, Jg. 10 (2011-07-25), S. 4088-4104 |
Veröffentlichung: | American Chemical Society (ACS), 2011 |
Medientyp: | unknown |
ISSN: | 1535-3907 (print) ; 1535-3893 (print) |
DOI: | 10.1021/pr2002726 |
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