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Phospho-iTRAQ: assessing isobaric labels for the large-scale study of phosphopeptide stoichiometry

Pieter Glibert, Paulien Meert, Katleen Van Steendam, Filip Van Nieuwerburgh UGent, Dieter De Coninck, Lennart Martens UGent, Maarten Dhaenens UGent and Dieter Deforce UGent (2015) JOURNAL OF PROTEOME RESEARCH. 14(2). p.839-849
abstract
The ability to distinguish between phosphopeptides of high and low stoichiometry is essential to discover the true extent of protein phosphorylation. We here extend the strategy whereby a peptide sample is briefly split in two identical parts and differentially labeled preceding the phosphatase treatment of one part. Our use of isobaric tags for relative and absolute quantitation (iTRAQ) marks the first time that isobaric tags have been applied for the large-scale analysis of phosphopeptides. Our Phospho-iTRAQ method focuses on the unmodified counterparts of phosphorylated peptides, which thus circumvents the ionization, fragmentation, and phospho-enrichment difficulties that hamper quantitation of stoichiometry in most common phosphoproteomics methods. Since iTRAQ enables multiplexing, simultaneous (phospho)proteome comparison between internal replicates and multiple samples is possible. The technique was validated on multiple instrument platforms by adding internal standards of high stoichiometry to a complex lysate of control and EGF-stimulated HeLa cells. To demonstrate the flexibility of PhosphoiTRAQ with regards to the experimental setup, the proteome coverage was extended through gel fractionation, while an internal replicate measurement created more stringent data analysis opportunities. The latest developments in MS instrumentation promise to further increase the resolution of the stoichiometric measurement of Phospho-iTRAQ in the future. The data have been deposited to the ProteomeXchange with identifier PXD001574.
Please use this url to cite or link to this publication:
author
organization
year
type
journalArticle (original)
publication status
published
subject
keyword
dephosphotylation, iTRAQ, stoichomtery, phosphoproteome, threshold calculation, in-gel Phospho-iTRAQ, MASS-SPECTROMETRY, QUANTITATIVE PROTEOMICS, PROTEIN-PHOSPHORYLATION, POSTTRANSLATIONAL MODIFICATIONS, QUANTIFICATION, PHOSPHOPROTEOMICS, STRATEGY, PEPTIDE, MITOSIS, SITES
journal title
JOURNAL OF PROTEOME RESEARCH
J. Proteome Res.
volume
14
issue
2
pages
839 - 849
Web of Science type
Article
Web of Science id
000349276400024
JCR category
BIOCHEMICAL RESEARCH METHODS
JCR impact factor
4.173 (2015)
JCR rank
12/77 (2015)
JCR quartile
1 (2015)
ISSN
1535-3893
DOI
10.1021/pr500889v
project
Bioinformatics: from nucleotids to networks (N2N)
language
English
UGent publication?
yes
classification
A1
copyright statement
I have transferred the copyright for this publication to the publisher
id
5801431
handle
http://hdl.handle.net/1854/LU-5801431
date created
2015-01-09 12:54:01
date last changed
2016-12-19 15:41:55
@article{5801431,
  abstract     = {The ability to distinguish between phosphopeptides of high and low stoichiometry is essential to discover the true extent of protein phosphorylation. We here extend the strategy whereby a peptide sample is briefly split in two identical parts and differentially labeled preceding the phosphatase treatment of one part. Our use of isobaric tags for relative and absolute quantitation (iTRAQ) marks the first time that isobaric tags have been applied for the large-scale analysis of phosphopeptides. Our Phospho-iTRAQ method focuses on the unmodified counterparts of phosphorylated peptides, which thus circumvents the ionization, fragmentation, and phospho-enrichment difficulties that hamper quantitation of stoichiometry in most common phosphoproteomics methods. Since iTRAQ enables multiplexing, simultaneous (phospho)proteome comparison between internal replicates and multiple samples is possible. The technique was validated on multiple instrument platforms by adding internal standards of high stoichiometry to a complex lysate of control and EGF-stimulated HeLa cells. To demonstrate the flexibility of PhosphoiTRAQ with regards to the experimental setup, the proteome coverage was extended through gel fractionation, while an internal replicate measurement created more stringent data analysis opportunities. The latest developments in MS instrumentation promise to further increase the resolution of the stoichiometric measurement of Phospho-iTRAQ in the future. The data have been deposited to the ProteomeXchange with identifier PXD001574.},
  author       = {Glibert, Pieter and Meert, Paulien and Van Steendam, Katleen and Van Nieuwerburgh, Filip and De Coninck, Dieter and Martens, Lennart and Dhaenens, Maarten and Deforce, Dieter},
  issn         = {1535-3893},
  journal      = {JOURNAL OF PROTEOME RESEARCH},
  keyword      = {dephosphotylation,iTRAQ,stoichomtery,phosphoproteome,threshold calculation,in-gel Phospho-iTRAQ,MASS-SPECTROMETRY,QUANTITATIVE PROTEOMICS,PROTEIN-PHOSPHORYLATION,POSTTRANSLATIONAL MODIFICATIONS,QUANTIFICATION,PHOSPHOPROTEOMICS,STRATEGY,PEPTIDE,MITOSIS,SITES},
  language     = {eng},
  number       = {2},
  pages        = {839--849},
  title        = {Phospho-iTRAQ: assessing isobaric labels for the large-scale study of phosphopeptide stoichiometry},
  url          = {http://dx.doi.org/10.1021/pr500889v},
  volume       = {14},
  year         = {2015},
}

Chicago
Glibert, Pieter, Paulien Meert, Katleen Van Steendam, Filip Van Nieuwerburgh, Dieter De Coninck, Lennart Martens, Maarten Dhaenens, and Dieter Deforce. 2015. “Phospho-iTRAQ: Assessing Isobaric Labels for the Large-scale Study of Phosphopeptide Stoichiometry.” Journal of Proteome Research 14 (2): 839–849.
APA
Glibert, P., Meert, P., Van Steendam, K., Van Nieuwerburgh, F., De Coninck, D., Martens, L., Dhaenens, M., et al. (2015). Phospho-iTRAQ: assessing isobaric labels for the large-scale study of phosphopeptide stoichiometry. JOURNAL OF PROTEOME RESEARCH, 14(2), 839–849.
Vancouver
1.
Glibert P, Meert P, Van Steendam K, Van Nieuwerburgh F, De Coninck D, Martens L, et al. Phospho-iTRAQ: assessing isobaric labels for the large-scale study of phosphopeptide stoichiometry. JOURNAL OF PROTEOME RESEARCH. 2015;14(2):839–49.
MLA
Glibert, Pieter, Paulien Meert, Katleen Van Steendam, et al. “Phospho-iTRAQ: Assessing Isobaric Labels for the Large-scale Study of Phosphopeptide Stoichiometry.” JOURNAL OF PROTEOME RESEARCH 14.2 (2015): 839–849. Print.