Open AccessComparative analysis of Erk phosphorylation suggests a mixed strategy for measuring phospho‐form distributions
Author(s) -
Prabakaran Sudhakaran,
Everley Robert A,
Landrieu Isabelle,
Wieruszeski JeanMichel,
Lippens Guy,
Steen Hanno,
Gunawardena Jeremy
Publication year - 2011
Publication title -
molecular systems biology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 8.523
H-Index - 148
ISSN - 1744-4292
DOI - 10.1038/msb.2011.15
Subject(s) - phosphorylation , blot , biology , kinase , protein phosphorylation , nuclear magnetic resonance spectroscopy , peptide , mass spectrometry , mapk/erk pathway , biochemistry , protein kinase a , chemistry , chromatography , gene , organic chemistry
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.