# Comparative Analysis of Erk Phosphorylation Suggests a Mixed Strategy for Measuring Phospho-Form Distributions

 Title: Comparative Analysis of Erk Phosphorylation Suggests a Mixed Strategy for Measuring Phospho-Form Distributions Author: Prabakaran, Sudhakaran; Everley, Robert A; Landrieu, Isabelle; Wieruszeski, Jean-Michel; Lippens, Guy; Steen, Hanno; Gunawardena, Jeremy H. Note: Order does not necessarily reflect citation order of authors. Citation: Prabakaran, Sudhakaran, Robert A. Everley, Isabelle Landrieu, Jean-Michel Wieruszeski, Guy Lippens, Hanno Steen, and Jeremy Gunawardena. 2011. Comparative analysis of Erk phosphorylation suggests a mixed strategy for measuring phospho-form distributions. Molecular Systems Biology 7: 482. Full Text & Related Files: 3097084.pdf (1.432Mb; PDF) 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 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. Published Version: 10.1038/msb.2011.15 Other Sources: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3097084/pdf/ Terms of Use: This article is made available under the terms and conditions applicable to Other Posted Material, as set forth at http://nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of-use#LAA Citable link to this page: http://nrs.harvard.edu/urn-3:HUL.InstRepos:5360053 Downloads of this work: