A COFRADIC Protocol To Study Protein Ubiquitination

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Abstract

Here, we apply the COmbined FRActional DIagonal Chromatography (COFRADIC) technology to enrich for ubiquitinated peptides and to identify sites of ubiquitination by mass spectrometry. Our technology bypasses the need to overexpress tagged variants of ubiquitin and the use of sequence-biased antibodies recognizing ubiquitin remnants. In brief, all protein primary amino groups are blocked by chemical acetylation, after which ubiquitin chains are proteolytically and specifically removed by the catalytic core domain of the USP2 deubiquitinase (USP2cc). Because USP2cc cleaves the isopeptidyl bond between the ubiquitin C-terminus and the ε-amino group of the ubiquitinated lysine, this enzyme reintroduces primary ε-amino groups in proteins. These amino groups are then chemically modified with a handle that allows specific isolation of ubiquitinated peptides during subsequent COFRADIC chromatographic runs. This method led to the identification of over 7500 endogenous ubiquitination sites in more than 3300 different proteins in a native human Jurkat cell lysate.

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