Prabakaran, SudhakaranEverley, Robert ALandrieu, IsabelleWieruszeski, Jean-MichelLippens, GuySteen, HannoGunawardena, Jeremy2011-12-052011Prabakaran, 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: 482.1744-4292http://nrs.harvard.edu/urn-3:HUL.InstRepos:5360053The 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.en-USmass spectrometrymultisite phosphorylationNMRphospho-form distributionphosphospecific antibodiesJournal Article2011-12-0510.1038/msb.2011.15