A COFRADIC Protocol To Study Protein Ubiquitination

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.