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Prabakaran, Sudhakaran

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Prabakaran

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Sudhakaran

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Prabakaran, Sudhakaran
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    Comparative Analysis of Erk Phosphorylation Suggests a Mixed Strategy for Measuring Phospho-Form Distributions
    (Nature Publishing Group, 2011) Prabakaran, Sudhakaran; Everley, Robert A; Landrieu, Isabelle; Wieruszeski, Jean-Michel; Lippens, Guy; Steen, Hanno; Gunawardena, Jeremy
    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.