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A COFRADIC protocol to study protein ubiquitination

Elisabeth Stes, Mathias Laga UGent, Alan Walton, Noortje Samyn, Evy Timmerman UGent, Ive De Smet UGent, Sofie Goormachtig UGent and Kris Gevaert UGent (2014) JOURNAL OF PROTEOME RESEARCH. 13(6). p.3107-3113
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.
Please use this url to cite or link to this publication:
author
organization
year
type
journalArticle (original)
publication status
published
subject
keyword
protein ubiquitination, DEGRADATION, USP2cc, QUANTIFICATION, MASS-SPECTROMETRY, IDENTIFICATION, LIGASE, SITES, COFRADIC, N-TERMINAL PEPTIDES, FRACTIONAL DIAGONAL CHROMATOGRAPHY
journal title
JOURNAL OF PROTEOME RESEARCH
J. Proteome Res.
volume
13
issue
6
pages
3107 - 3113
Web of Science type
Article
Web of Science id
000337074500035
JCR category
BIOCHEMICAL RESEARCH METHODS
JCR impact factor
4.245 (2014)
JCR rank
14/79 (2014)
JCR quartile
1 (2014)
ISSN
1535-3893
DOI
10.1021/pr4012443
language
English
UGent publication?
yes
classification
A1
copyright statement
I have transferred the copyright for this publication to the publisher
id
5656277
handle
http://hdl.handle.net/1854/LU-5656277
date created
2014-07-14 15:36:06
date last changed
2016-12-19 15:45:58
@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},
}

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.