Peptide Array X-Linking (PAX): A New Peptide-Protein Identification Approach
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Okada-2012-Peptide array X-link.pdf
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3.19 MB
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Author(s)
Okada, Hirokazu
Uezu, Akiyoshi
Soderblom, Erik J.
Moseley, M. Arthur, III
Gertler, Frank
Soderling, Scott H.
Date Issued
May 2012
Journal
PLoS ONE
Publisher
Public Library of Science
Citation
Okada, Hirokazu et al. “Peptide Array X-Linking (PAX): A New Peptide-Protein Identification Approach.” Ed. Pontus Aspenstrom. PLoS ONE 7.5 (2012): e37035.
Version
Final published version
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.
MIT Department
Massachusetts Institute of Technology. Department of Biology
Koch Institute for Integrative Cancer Research at MIT
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DOI of Published Version
https://doi.org/10.1371/journal.pone.0037035
