title: Comparative analysis of Erk phosphorylation suggests a mixed strategy for measuring phospho-form distributions.
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.
authors.affiliation: Department of Systems Biology, Harvard Medical School, Boston, MA 02115, USA.
authors.forename: Sudhakaran
authors.initials: S
authors.lastname: Prabakaran
authors.forename: Robert A
authors.initials: RA
authors.lastname: Everley
authors.forename: Isabelle
authors.initials: I
authors.lastname: Landrieu
authors.forename: Jean-Michel
authors.initials: JM
authors.lastname: Wieruszeski
authors.forename: Guy
authors.initials: G
authors.lastname: Lippens
authors.forename: Hanno
authors.initials: H
authors.lastname: Steen
authors.forename: Jeremy
authors.initials: J
authors.lastname: Gunawardena
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D045163:Antibodies, Phospho-Specific / Q000378:metabolism; D015153:Blotting, Western / Q000379:methods; D002853:Chromatography, Liquid / Q000379:methods; D048370:MAP Kinase Kinase 2 / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D013058:Mass Spectrometry / Q000379:methods; D008969:Molecular Sequence Data; D010455:Peptides / Q000378:metabolism; D010766:Phosphorylation; D014981:Xenopus
chemical_list: D045163:Antibodies, Phospho-Specific; D010455:Peptides; D048370:MAP Kinase Kinase 2