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A COFRADIC protocol to study protein ubiquitination

Elisabeth Stes (UGent) , Mathias Laga (UGent) , Alan Walton (UGent) , Noortje Samyn (UGent) , Evy Timmerman (UGent) , Ive De Smet (UGent) , Sofie Goormachtig (UGent) and Kris Gevaert (UGent)
(2014) JOURNAL OF PROTEOME RESEARCH. 13(6). p.3107-3113
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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 epsilon-amino group of the ubiquitinated lysine, this enzyme reintroduces primary epsilon-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.
Keywords
protein ubiquitination, DEGRADATION, USP2cc, QUANTIFICATION, MASS-SPECTROMETRY, IDENTIFICATION, LIGASE, SITES, COFRADIC, N-TERMINAL PEPTIDES, FRACTIONAL DIAGONAL CHROMATOGRAPHY

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Chicago
Stes, Elisabeth, Mathias Laga, Alan Walton, Noortje Samyn, Evy Timmerman, Ive De Smet, Sofie Goormachtig, and Kris Gevaert. 2014. “A COFRADIC Protocol to Study Protein Ubiquitination.” Journal of Proteome Research 13 (6): 3107–3113.
APA
Stes, E., Laga, M., Walton, A., Samyn, N., Timmerman, E., De Smet, I., Goormachtig, S., et al. (2014). A COFRADIC protocol to study protein ubiquitination. JOURNAL OF PROTEOME RESEARCH, 13(6), 3107–3113.
Vancouver
1.
Stes E, Laga M, Walton A, Samyn N, Timmerman E, De Smet I, et al. A COFRADIC protocol to study protein ubiquitination. JOURNAL OF PROTEOME RESEARCH. 2014;13(6):3107–13.
MLA
Stes, Elisabeth, Mathias Laga, Alan Walton, et al. “A COFRADIC Protocol to Study Protein Ubiquitination.” JOURNAL OF PROTEOME RESEARCH 13.6 (2014): 3107–3113. Print.
@article{5656277,
  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 epsilon-amino group of the ubiquitinated lysine, this enzyme reintroduces primary epsilon-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.},
  author       = {Stes, Elisabeth and Laga, Mathias and Walton, Alan and Samyn, Noortje and Timmerman, Evy and De Smet, Ive and Goormachtig, Sofie and Gevaert, Kris},
  issn         = {1535-3893},
  journal      = {JOURNAL OF PROTEOME RESEARCH},
  keyword      = {protein ubiquitination,DEGRADATION,USP2cc,QUANTIFICATION,MASS-SPECTROMETRY,IDENTIFICATION,LIGASE,SITES,COFRADIC,N-TERMINAL PEPTIDES,FRACTIONAL DIAGONAL CHROMATOGRAPHY},
  language     = {eng},
  number       = {6},
  pages        = {3107--3113},
  title        = {A COFRADIC protocol to study protein ubiquitination},
  url          = {http://dx.doi.org/10.1021/pr4012443},
  volume       = {13},
  year         = {2014},
}

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