Enhanced detection of ubiquitin isopeptides using reductive methylation.
Authors
Chicooree, NavinConnolly, Yvonne
Tan, Chong-Teik
Malliri, Angeliki
Li, Yaoyong
Smith, Duncan L
Griffiths, John R
Affiliation
Paterson Institute for Cancer Research, University of Manchester, Manchester, M20 4BX, UK.Issue Date
2013-03
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Identification of ubiquitination (Ub) sites is of great interest due to the critical roles that the modification plays in cellular regulation. Current methods using mass spectrometry rely upon tryptic isopeptide diglycine tag generation followed by database searching. We present a novel approach to ubiquitin detection based upon the dimethyl labeling of isopeptide N-termini glycines. Ubiquitinated proteins were digested with trypsin and the resulting peptide mixture was derivatized using formaldehyde-D2 solution and sodium cyanoborohydride. The dimethylated peptide mixtures were next separated by liquid chromatography and analyzed on a quadrupole-TOF based mass spectrometer. Diagnostic b2' and a1' ions released from the isopeptide N-terminus upon collision-induced dissociation (CID) were used to spectrally improve the identification of ubiquitinated isopeptides. Proof of principle was established by application to a ubiquitinated protein tryptic digest spiked into a six-protein mix digest background. Extracted ion chromatograms of the a1' and b2' diagnostic product ions from the diglycine tag resulted in a significant reduction in signal complexity and demonstrated a selectivity towards the identification of diglycine branched isopeptides. The method was further shown to be capable of identifying diglycine isopeptides resulting from in-gel tryptic digests of ubiquitin enriched material from a His-Ub transfected cell line. We envisage that these ions may be utilized in global ubiquitination studies with post-acquisition MS/MS (or MSe) data interrogation on high resolution hybrid mass spectrometers. Figure ᅟCitation
Enhanced detection of ubiquitin isopeptides using reductive methylation. 2013, 24 (3):421-30 J Am Soc Mass SpectromJournal
Journal of the American Society for Mass SpectrometryDOI
10.1007/s13361-012-0538-0PubMed ID
23361369Type
ArticleLanguage
enISSN
1879-1123ae974a485f413a2113503eed53cd6c53
10.1007/s13361-012-0538-0
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