title: Motif-specific sampling of phosphoproteomes.
abstract: Phosphoproteomics, the targeted study of a subfraction of the proteome which is modified by phosphorylation, has become an indispensable tool to study cell signaling dynamics. We described a methodology that linked phosphoproteome and proteome analysis based on Ba2+ binding properties of amino acids. This technology selected motif-specific phosphopeptides independent of the system under analysis. MudPIT (Multidimensional Identification Technology) identified 1037 precipitated phosphopeptides from as little as 250 microg of proteins. To extend coverage of the phosphoproteome, we sampled the nuclear extract of HeLa cells with three values of Ba2+ ions molarity. The presence of more than 70% of identified phosphoproteins was further substantiated by their nonmodified peptides. Upon isoproterenol stimulation of HEK cells, we identified an increasing number of phosphoproteins from MAPK cascades and AKAP signaling hubs. We quantified changes in both protein and phosphorylation levels of 197 phosphoproteins including a critical kinase, MAPK1. Integration of differential phosphorylation of MAPK1 with knowledge bases constructed modules that correlated well with its role as node in cross-talk of canonical pathways.
authors.affiliation: Department of Chemical Physiology/Cell Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.
authors.forename: Cristian I
authors.initials: CI
authors.lastname: Ruse
authors.forename: Daniel B
authors.initials: DB
authors.lastname: McClatchy
authors.forename: Bingwen
authors.initials: B
authors.lastname: Lu
authors.forename: Daniel
authors.initials: D
authors.lastname: Cociorva
authors.forename: Akira
authors.initials: A
authors.lastname: Motoyama
authors.forename: Sung Kyu
authors.initials: SK
authors.lastname: Park
authors.forename: John R
authors.initials: JR
authors.lastname: Yates
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001464:Barium / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D010748:Phosphopeptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010750:Phosphoproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D020543:Proteome / Q000032:analysis*; D040901:Proteomics / Q000295:instrumentation / Q000379:methods*; D015398:Signal Transduction / Q000502:physiology
chemical_list: D010748:Phosphopeptides; D010750:Phosphoproteins; D020543:Proteome; D001464:Barium