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Enhanced Detection of Ubiquitin Isopeptides Using Reductive Methylation

Enhanced Detection of Ubiquitin Isopeptides Using Reductive Methylation

Navin Chicooree, Yvonne Connolly, Chong-Teik Tan, Angeliki Malliri, Yaoyong Li, Duncan L Smith, John R Griffiths

J Am Soc Mass Spectrom. 2013 Mar;24(3):421-30.

PMID: 23361369

Abstract:

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. ᅟ

Chemicals Related in the Paper:

Catalog Number	Product Name	Structure	CAS Number	Price
AP1664988	Formaldehyde-d2 solution		1664-98-8	Price

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