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

Overview

Journal Mol Syst Biol

Specialty Molecular Biology

Date 2011 Apr 14

PMID 21487401

Citations 24

Authors

Sudhakaran Prabakaran

Robert A Everley

Isabelle Landrieu

Jean-Michel Wieruszeski

Guy Lippens

Hanno Steen

Jeremy Gunawardena

Affiliations

Soon will be listed here.

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.

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