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

Pieter Glibert (UGent) , Paulien Meert (UGent) , Katleen Van Steendam (UGent) , Filip Van Nieuwerburgh (UGent) , Dieter De Coninck (UGent) , Lennart Martens (UGent) , Maarten Dhaenens (UGent) and Dieter Deforce (UGent)
(2015) JOURNAL OF PROTEOME RESEARCH. 14(2). p.839-849
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Bioinformatics: from nucleotids to networks (N2N)
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.
Keywords
dephosphotylation, iTRAQ, stoichomtery, phosphoproteome, threshold calculation, in-gel Phospho-iTRAQ, MASS-SPECTROMETRY, QUANTITATIVE PROTEOMICS, PROTEIN-PHOSPHORYLATION, POSTTRANSLATIONAL MODIFICATIONS, QUANTIFICATION, PHOSPHOPROTEOMICS, STRATEGY, PEPTIDE, MITOSIS, SITES

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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.
@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},
}

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