Peptide Array X-Linking (PAX): A New Peptide-Protein Identification Approach
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 ami...
| Main Authors: | , , , , , |
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| Other Authors: | |
| Format: | Article |
| Language: | en_US |
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Public Library of Science
2012
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| Online Access: | http://hdl.handle.net/1721.1/71732 https://orcid.org/0000-0003-3214-4554 |
| _version_ | 1811081422178353152 |
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| author | Okada, Hirokazu Uezu, Akiyoshi Soderblom, Erik J. Moseley, M. Arthur, III Gertler, Frank Soderling, Scott H. |
| author2 | Massachusetts Institute of Technology. Department of Biology |
| author_facet | Massachusetts Institute of Technology. Department of Biology Okada, Hirokazu Uezu, Akiyoshi Soderblom, Erik J. Moseley, M. Arthur, III Gertler, Frank Soderling, Scott H. |
| author_sort | Okada, Hirokazu |
| collection | MIT |
| description | 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. |
| first_indexed | 2024-09-23T11:46:25Z |
| format | Article |
| id | mit-1721.1/71732 |
| institution | Massachusetts Institute of Technology |
| language | en_US |
| last_indexed | 2024-09-23T11:46:25Z |
| publishDate | 2012 |
| publisher | Public Library of Science |
| record_format | dspace |
| spelling | mit-1721.1/717322022-09-27T21:48:48Z Peptide Array X-Linking (PAX): A New Peptide-Protein Identification Approach Okada, Hirokazu Uezu, Akiyoshi Soderblom, Erik J. Moseley, M. Arthur, III Gertler, Frank Soderling, Scott H. Massachusetts Institute of Technology. Department of Biology Koch Institute for Integrative Cancer Research at MIT Gertler, Frank Gertler, Frank 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. National Institutes of Health (U.S.) (Grant R21-CA-140030-01) 2012-07-20T17:45:55Z 2012-07-20T17:45:55Z 2012-05 2012-01 Article http://purl.org/eprint/type/JournalArticle 1932-6203 http://hdl.handle.net/1721.1/71732 Okada, Hirokazu et al. “Peptide Array X-Linking (PAX): A New Peptide-Protein Identification Approach.” Ed. Pontus Aspenstrom. PLoS ONE 7.5 (2012): e37035. https://orcid.org/0000-0003-3214-4554 en_US http://dx.doi.org/10.1371/journal.pone.0037035 PLoS ONE Creative Commons Attribution http://creativecommons.org/licenses/by/2.5/ application/pdf Public Library of Science PLoS |
| spellingShingle | Okada, Hirokazu Uezu, Akiyoshi Soderblom, Erik J. Moseley, M. Arthur, III Gertler, Frank Soderling, Scott H. Peptide Array X-Linking (PAX): A New Peptide-Protein Identification Approach |
| title | Peptide Array X-Linking (PAX): A New Peptide-Protein Identification Approach |
| title_full | Peptide Array X-Linking (PAX): A New Peptide-Protein Identification Approach |
| title_fullStr | Peptide Array X-Linking (PAX): A New Peptide-Protein Identification Approach |
| title_full_unstemmed | Peptide Array X-Linking (PAX): A New Peptide-Protein Identification Approach |
| title_short | Peptide Array X-Linking (PAX): A New Peptide-Protein Identification Approach |
| title_sort | peptide array x linking pax a new peptide protein identification approach |
| url | http://hdl.handle.net/1721.1/71732 https://orcid.org/0000-0003-3214-4554 |
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