Journal
MOLECULAR SYSTEMS BIOLOGY
Volume 7, Issue -, Pages -Publisher
NATURE PUBLISHING GROUP
DOI: 10.1038/msb.2011.15
Keywords
mass spectrometry; multisite phosphorylation; NMR; phospho-form distribution; phosphospecific antibodies
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Funding
- NIH [R01-GM081578]
- NRSAT32 [HL076115]
- TGE RMN THC (France) [FR-3050]
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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 2(n) 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. Molecular Systems Biology 7: 482; published online 12 April 2011; doi:10.1038/msb.2011.15
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