title: Peptide array X-linking (PAX): a new peptide-protein identification approach.
abstract: Many protein interaction domains bind short peptides based on canonical sequence consensus motifs. Here we report the development of a peptide array-based proteomics tool to identify proteins directly interacting with ligand peptides from cell lysates. Array-formatted bait peptides containing an amino acid-derived cross-linker are photo-induced to crosslink with interacting proteins from lysates of interest. Indirect associations are removed by high stringency washes under denaturing conditions. Covalently trapped proteins are subsequently identified by LC-MS/MS and screened by cluster analysis and domain scanning. We apply this methodology to peptides with different proline-containing consensus sequences and show successful identifications from brain lysates of known and novel proteins containing polyproline motif-binding domains such as EH, EVH1, SH3, WW domains. These results suggest the capacity of arrayed peptide ligands to capture and subsequently identify proteins by mass spectrometry is relatively broad and robust. Additionally, the approach is rapid and applicable to cell or tissue fractions from any source, making the approach a flexible tool for initial protein-protein interaction discovery.
authors.affiliation: Department of Cell Biology, Duke University Medical School, Durham, North Carolina, United States of America.
authors.forename: Hirokazu
authors.initials: H
authors.lastname: Okada
authors.forename: Akiyoshi
authors.initials: A
authors.lastname: Uezu
authors.forename: Erik J
authors.initials: EJ
authors.lastname: Soderblom
authors.forename: M Arthur
authors.initials: MA
authors.lastname: Moseley
authors.forename: Frank B
authors.initials: FB
authors.lastname: Gertler
authors.forename: Scott H
authors.initials: SH
authors.lastname: Soderling
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001923:Brain Chemistry; D003432:Cross-Linking Reagents; D047908:Intracellular Signaling Peptides and Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019151:Peptide Library; D054730:Protein Interaction Domains and Motifs; D040901:Proteomics / Q000379:methods*; D053719:Tandem Mass Spectrometry; D051300:Wiskott-Aldrich Syndrome Protein Family / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D003432:Cross-Linking Reagents; D047908:Intracellular Signaling Peptides and Proteins; D009419:Nerve Tissue Proteins; D019151:Peptide Library; D051300:Wiskott-Aldrich Syndrome Protein Family