The functional impact of multisite protein phosphorylation can depend on both the numbers and the positions of phosphorylated sites—the global pattern of phosphorylation or 'phospho-form'—giving biological systems profound capabilities for dynamic information processing. A central problem in quantitative systems biology, therefore, is to measure the 'phospho-form distribution': the relative amount of each of the 2n phospho-forms of a protein with n-phosphorylation sites. We compared four potential methods—western blots with phospho-specific antibodies, peptide-based liquid chromatography (LC) and mass spectrometry (MS; pepMS), protein-based LC/MS (proMS) and nuclear magnetic resonance spectroscopy (NMR)—on differentially phosphorylated samples of the well-studied mitogen-activated protein kinase Erk2, with two phosphorylation sites. The MS methods were quantitatively consistent with each other and with NMR to within 10\%, but western blots, while highly sensitive, showed significant discrepancies with MS. NMR also uncovered two additional phosphorylations, for which a combination of pepMS and proMS yielded an estimate of the 16-member phospho-form distribution. This combined MS strategy provides an optimal mixture of accuracy and coverage for quantifying distributions, but positional isomers remain a challenging problem.
%0 Journal Article
%1 Prabakaran2011Comparative
%A Prabakaran, Sudhakaran
%A Everley, Robert A.
%A Landrieu, Isabelle
%A Wieruszeski, Jean-Michel
%A Lippens, Guy
%A Steen, Hanno
%A Gunawardena, Jeremy
%D 2011
%I Nature Publishing Group
%J Molecular Systems Biology
%K experimental-technique phosphorylation
%R 10.1038/msb.2011.15
%T Comparative analysis of Erk phosphorylation suggests a mixed strategy for measuring phospho-form distributions
%U http://dx.doi.org/10.1038/msb.2011.15
%V 7
%X The functional impact of multisite protein phosphorylation can depend on both the numbers and the positions of phosphorylated sites—the global pattern of phosphorylation or 'phospho-form'—giving biological systems profound capabilities for dynamic information processing. A central problem in quantitative systems biology, therefore, is to measure the 'phospho-form distribution': the relative amount of each of the 2n phospho-forms of a protein with n-phosphorylation sites. We compared four potential methods—western blots with phospho-specific antibodies, peptide-based liquid chromatography (LC) and mass spectrometry (MS; pepMS), protein-based LC/MS (proMS) and nuclear magnetic resonance spectroscopy (NMR)—on differentially phosphorylated samples of the well-studied mitogen-activated protein kinase Erk2, with two phosphorylation sites. The MS methods were quantitatively consistent with each other and with NMR to within 10\%, but western blots, while highly sensitive, showed significant discrepancies with MS. NMR also uncovered two additional phosphorylations, for which a combination of pepMS and proMS yielded an estimate of the 16-member phospho-form distribution. This combined MS strategy provides an optimal mixture of accuracy and coverage for quantifying distributions, but positional isomers remain a challenging problem.
@article{Prabakaran2011Comparative,
abstract = {The functional impact of multisite protein phosphorylation can depend on both the numbers and the positions of phosphorylated sites—the global pattern of phosphorylation or 'phospho-form'—giving biological systems profound capabilities for dynamic information processing. A central problem in quantitative systems biology, therefore, is to measure the 'phospho-form distribution': the relative amount of each of the 2n phospho-forms of a protein with n-phosphorylation sites. We compared four potential methods—western blots with phospho-specific antibodies, peptide-based liquid chromatography ({LC}) and mass spectrometry ({MS}; {pepMS}), protein-based {LC}/{MS} ({proMS}) and nuclear magnetic resonance spectroscopy ({NMR})—on differentially phosphorylated samples of the well-studied mitogen-activated protein kinase Erk2, with two phosphorylation sites. The {MS} methods were quantitatively consistent with each other and with {NMR} to within 10\%, but western blots, while highly sensitive, showed significant discrepancies with {MS}. {NMR} also uncovered two additional phosphorylations, for which a combination of {pepMS} and {proMS} yielded an estimate of the 16-member phospho-form distribution. This combined {MS} strategy provides an optimal mixture of accuracy and coverage for quantifying distributions, but positional isomers remain a challenging problem.},
added-at = {2018-12-02T16:09:07.000+0100},
author = {Prabakaran, Sudhakaran and Everley, Robert A. and Landrieu, Isabelle and Wieruszeski, Jean-Michel and Lippens, Guy and Steen, Hanno and Gunawardena, Jeremy},
biburl = {https://www.bibsonomy.org/bibtex/2f2e6841fce6fa812cfc2f3769efd0da9/karthikraman},
citeulike-article-id = {9149362},
citeulike-linkout-0 = {http://dx.doi.org/10.1038/msb.2011.15},
citeulike-linkout-1 = {http://dx.doi.org/10.1038/msb201115},
day = 12,
doi = {10.1038/msb.2011.15},
interhash = {c0b405354a9ef22820719243b3b41666},
intrahash = {f2e6841fce6fa812cfc2f3769efd0da9},
journal = {Molecular Systems Biology},
keywords = {experimental-technique phosphorylation},
month = apr,
posted-at = {2011-04-29 09:36:29},
priority = {2},
publisher = {Nature Publishing Group},
timestamp = {2018-12-02T16:09:07.000+0100},
title = {Comparative analysis of Erk phosphorylation suggests a mixed strategy for measuring phospho-form distributions},
url = {http://dx.doi.org/10.1038/msb.2011.15},
volume = 7,
year = 2011
}