Mass spectrometry detection of histidine phosphorylation on NM23-H1.
Phosphorylation is a ubiquitous protein post-translational modification that is intimately involved in most aspects of cellular regulation. Currently, most proteomic analyses are performed with phosphorylation searches for serine, threonine, and tyrosine modifications, as the phosphorylated residues of histidine and aspartic acid are acid labile and thus undetectable with most proteomic methodologies. Here, we present a novel buffer system to show histidine phosphorylation of NM23-H1, the product of the first identified putative human metastasis suppressor gene (NME1), which catalyzes the transfer of the γ-phosphate from nucleoside triphosphates to nucleoside diphosphates. On the basis of a pH titration of LC elution buffers and MS/MS identification, recombinant NM23-H1 subjected to autophosphorylation was shown to contain phosphorylated histidine at residue 118 at pH 5 and 6, with each level giving over 75% peptide coverage for identification. The solvent system presented permits the detection of all five possible phosphorylation moieties. Application of histidine and aspartic acid phosphorylation modifications to proteomic analyses will significantly advance the understanding of phosphorylation relay signaling in cellular regulation, including elucidation of the role of NM23-H1 in metastasis.
Department of Environmental Medicine, University of Rochester Medical Center, Rochester, New York 14642, USA.
John D
JD
Lapek
Gregory
G
Tombline
Alan E
AE
Friedman
D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000737:chemistry / Q000378:metabolism; D002853:Chromatography, Liquid; D006639:Histidine / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D054778:NM23 Nucleoside Diphosphate Kinases / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D040901:Proteomics / Q000379:methods; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D053719:Tandem Mass Spectrometry / Q000379:methods*
D054778:NM23 Nucleoside Diphosphate Kinases; D011994:Recombinant Proteins; D001224:Aspartic Acid; D006639:Histidine; C517938:NME1 protein, human