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Comparative analysis of Erk phosphorylation suggests a mixed strategy for measuring phospho-form distributions.

Published version
Peer-reviewed

Type

Article

Change log

Authors

Prabakaran, Sudhakaran  ORCID logo  https://orcid.org/0000-0002-6527-1085
Everley, Robert A 
Landrieu, Isabelle 
Wieruszeski, Jean-Michel 
Lippens, Guy 

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.

Description

Keywords

Amino Acid Sequence, Animals, Antibodies, Phospho-Specific, Blotting, Western, Chromatography, Liquid, MAP Kinase Kinase 2, Magnetic Resonance Spectroscopy, Mass Spectrometry, Molecular Sequence Data, Peptides, Phosphorylation, Xenopus

Journal Title

Mol Syst Biol

Conference Name

Journal ISSN

1744-4292
1744-4292

Volume Title

7

Publisher

Springer Science and Business Media LLC