title: Proteomic screening method for phosphopeptide motif binding proteins using peptide libraries.
abstract: Phosphopeptide binding domains mediate the directed and localized assembly of protein complexes essential to intracellular kinase signaling. To identify phosphopeptide binding proteins, we developed a proteomic screening method using immobilized partially degenerate phosphopeptide mixtures combined with SILAC and microcapillary LC-MS/MS. The method was used to identify proteins that specifically bound to phosphorylated peptide library affinity matrices, including pTyr, and the motifs pSer/pThr-Pro, pSer/pThr-X-X-X-pSer/pThr, pSer/pThr-Glu/Asp, or pSer/pThr-pSer/pThr in degenerate sequence contexts. Heavy and light SILAC lysates were applied to columns containing these phosphorylated and nonphosphorylated (control) peptide libraries respectively, and bound proteins were eluted, combined, digested, and analyzed by LC-MS/MS using a hybrid quadrupole-TOF mass spectrometer. Heavy/light peptide ion ratios were calculated, and peptides that yielded ratios greater than ∼3:1 were considered as being from potential phosphopeptide binding proteins since this ratio represents the lowest ratio from a known positive control. Many of those identified were known phosphopeptide-binding proteins, including the SH2 domain containing p85 subunit of PI3K bound to pTyr, 14-3-3 bound to pSer/pThr-Asp/Glu, polo-box domain containing PLK1 and Pin1 bound to pSer/pThr-Pro, and pyruvate kinase M2 binding to pTyr. Approximately half of the hits identified by the peptide library screens were novel. Protein domain enrichment analysis revealed that most pTyr hits contain SH2 domains, as expected, and to a lesser extent SH3, C1, STAT, Tyr phosphatase, Pkinase, C2, and PH domains; however, pSer/pThr motifs did not reveal enriched domains across hits.
authors.affiliation: Department of Systems Biology, Harvard Medical School, Boston, Massachusetts 02115, United States.
authors.forename: Heather R
authors.initials: HR
authors.lastname: Christofk
authors.forename: Ning
authors.initials: N
authors.lastname: Wu
authors.forename: Lewis C
authors.initials: LC
authors.lastname: Cantley
authors.forename: John M
authors.initials: JM
authors.lastname: Asara
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D006367:HeLa Cells; D006801:Humans; D007553:Isotope Labeling; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D019151:Peptide Library; D019606:Phosphoamino Acids / Q000737:chemistry / Q000378:metabolism; D010748:Phosphopeptides / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D011506:Proteins / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*; D018909:src Homology Domains
chemical_list: D019151:Peptide Library; D019606:Phosphoamino Acids; D010748:Phosphopeptides; D011506:Proteins