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Peptide array X-linking (PAX): a new peptide-protein identification approach.

Okada H, Uezu A, Soderblom EJ, Moseley MA, Gertler FB, Soderling SH - PLoS ONE (2012)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Duke University Medical School, Durham, North Carolina, United States of America.

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.

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PAX identifies novel interacting proteins with WAVE1.(A) Identification of ligands for a WAVE1 derived peptide. MS/MS data for WAVE1 trapped proteins from brain lysate were combined with those for control, SOS1, Stonin2, Epsin1 and mGluR5 and subjected to cluster analysis as in Fig. 3A. Vasp and Gas7 are known to interact with other members of the WAVE/WASP family proteins. (B) Protein interaction domain filtering. Enah and Amph, in addition to Vasp and Gas7, were identified as WAVE1 peptide-interacting proteins. (C) Only 3 of the 13 proteins identified in (B) were identified as abundant proteins using label-free LC/MS/MS quantification of mouse brain lysates. (D) WAVE1 peptide interactome. PAX identified high confidence interactions (red dotted arrows) with WAVE1. The previously known interactions are indicated with black arrows. The WAVE1 selective binding proteins contain domains (shown in red) that are expected to interact with the WAVE1 polyproline peptide (see Table 1). (E–F) Co-immunoprecipitation assays with full-length WAVE1 and (E) Amph or (F) Gas7. (G) GST pulldown of Enah using the proline peptide bait of WAVE1. (H) Co-immunoprecipitation of GFP-Enah with full-length WAVE1 and WAVE1 lacking the bait proline peptide. (I) Physiological interaction between WAVE1 and Enah. Reciprocal co-immunoprecipitation experiments were performed using rat brain extract (RBE). The precipitates were analyzed by Western blotting. (J) Co-localization of WAVE1 and Enah in fibroblast cells. Immunostaining of endogenous WAVE1 showed co-localization with Enah in MVD7 cells expressing GFP-tagged Enah.
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pone-0037035-g004: PAX identifies novel interacting proteins with WAVE1.(A) Identification of ligands for a WAVE1 derived peptide. MS/MS data for WAVE1 trapped proteins from brain lysate were combined with those for control, SOS1, Stonin2, Epsin1 and mGluR5 and subjected to cluster analysis as in Fig. 3A. Vasp and Gas7 are known to interact with other members of the WAVE/WASP family proteins. (B) Protein interaction domain filtering. Enah and Amph, in addition to Vasp and Gas7, were identified as WAVE1 peptide-interacting proteins. (C) Only 3 of the 13 proteins identified in (B) were identified as abundant proteins using label-free LC/MS/MS quantification of mouse brain lysates. (D) WAVE1 peptide interactome. PAX identified high confidence interactions (red dotted arrows) with WAVE1. The previously known interactions are indicated with black arrows. The WAVE1 selective binding proteins contain domains (shown in red) that are expected to interact with the WAVE1 polyproline peptide (see Table 1). (E–F) Co-immunoprecipitation assays with full-length WAVE1 and (E) Amph or (F) Gas7. (G) GST pulldown of Enah using the proline peptide bait of WAVE1. (H) Co-immunoprecipitation of GFP-Enah with full-length WAVE1 and WAVE1 lacking the bait proline peptide. (I) Physiological interaction between WAVE1 and Enah. Reciprocal co-immunoprecipitation experiments were performed using rat brain extract (RBE). The precipitates were analyzed by Western blotting. (J) Co-localization of WAVE1 and Enah in fibroblast cells. Immunostaining of endogenous WAVE1 showed co-localization with Enah in MVD7 cells expressing GFP-tagged Enah.

Mentions: We next applied the PAX and subsequent data analyses to the proline-rich sequence of WAVE1, a member of the Arp2/3-dependent actin nucleation promoting factors, to identify novel interactions from mouse brain lysates. To apply the clustering analysis, we combined MS data for WAVE1 with the previous MS data obtained for SOS1, Stonin2, Epsin1 and mGluR5 to elucidate WAVE1-specific protein cluster (Fig. 4A). The subsequent bioinformatics analysis identified no previously known interactors with WAVE1 but 2 plausible interactors, Vasodilator-stimulated phosphoprotein (Vasp) and Growth arrest-specific protein 7 (Gas7), that both have been revealed to interact with highly homologous family members of WAVE1 (WAS and WAVE2).


Peptide array X-linking (PAX): a new peptide-protein identification approach.

Okada H, Uezu A, Soderblom EJ, Moseley MA, Gertler FB, Soderling SH - PLoS ONE (2012)

PAX identifies novel interacting proteins with WAVE1.(A) Identification of ligands for a WAVE1 derived peptide. MS/MS data for WAVE1 trapped proteins from brain lysate were combined with those for control, SOS1, Stonin2, Epsin1 and mGluR5 and subjected to cluster analysis as in Fig. 3A. Vasp and Gas7 are known to interact with other members of the WAVE/WASP family proteins. (B) Protein interaction domain filtering. Enah and Amph, in addition to Vasp and Gas7, were identified as WAVE1 peptide-interacting proteins. (C) Only 3 of the 13 proteins identified in (B) were identified as abundant proteins using label-free LC/MS/MS quantification of mouse brain lysates. (D) WAVE1 peptide interactome. PAX identified high confidence interactions (red dotted arrows) with WAVE1. The previously known interactions are indicated with black arrows. The WAVE1 selective binding proteins contain domains (shown in red) that are expected to interact with the WAVE1 polyproline peptide (see Table 1). (E–F) Co-immunoprecipitation assays with full-length WAVE1 and (E) Amph or (F) Gas7. (G) GST pulldown of Enah using the proline peptide bait of WAVE1. (H) Co-immunoprecipitation of GFP-Enah with full-length WAVE1 and WAVE1 lacking the bait proline peptide. (I) Physiological interaction between WAVE1 and Enah. Reciprocal co-immunoprecipitation experiments were performed using rat brain extract (RBE). The precipitates were analyzed by Western blotting. (J) Co-localization of WAVE1 and Enah in fibroblast cells. Immunostaining of endogenous WAVE1 showed co-localization with Enah in MVD7 cells expressing GFP-tagged Enah.
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Related In: Results  -  Collection

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pone-0037035-g004: PAX identifies novel interacting proteins with WAVE1.(A) Identification of ligands for a WAVE1 derived peptide. MS/MS data for WAVE1 trapped proteins from brain lysate were combined with those for control, SOS1, Stonin2, Epsin1 and mGluR5 and subjected to cluster analysis as in Fig. 3A. Vasp and Gas7 are known to interact with other members of the WAVE/WASP family proteins. (B) Protein interaction domain filtering. Enah and Amph, in addition to Vasp and Gas7, were identified as WAVE1 peptide-interacting proteins. (C) Only 3 of the 13 proteins identified in (B) were identified as abundant proteins using label-free LC/MS/MS quantification of mouse brain lysates. (D) WAVE1 peptide interactome. PAX identified high confidence interactions (red dotted arrows) with WAVE1. The previously known interactions are indicated with black arrows. The WAVE1 selective binding proteins contain domains (shown in red) that are expected to interact with the WAVE1 polyproline peptide (see Table 1). (E–F) Co-immunoprecipitation assays with full-length WAVE1 and (E) Amph or (F) Gas7. (G) GST pulldown of Enah using the proline peptide bait of WAVE1. (H) Co-immunoprecipitation of GFP-Enah with full-length WAVE1 and WAVE1 lacking the bait proline peptide. (I) Physiological interaction between WAVE1 and Enah. Reciprocal co-immunoprecipitation experiments were performed using rat brain extract (RBE). The precipitates were analyzed by Western blotting. (J) Co-localization of WAVE1 and Enah in fibroblast cells. Immunostaining of endogenous WAVE1 showed co-localization with Enah in MVD7 cells expressing GFP-tagged Enah.
Mentions: We next applied the PAX and subsequent data analyses to the proline-rich sequence of WAVE1, a member of the Arp2/3-dependent actin nucleation promoting factors, to identify novel interactions from mouse brain lysates. To apply the clustering analysis, we combined MS data for WAVE1 with the previous MS data obtained for SOS1, Stonin2, Epsin1 and mGluR5 to elucidate WAVE1-specific protein cluster (Fig. 4A). The subsequent bioinformatics analysis identified no previously known interactors with WAVE1 but 2 plausible interactors, Vasodilator-stimulated phosphoprotein (Vasp) and Growth arrest-specific protein 7 (Gas7), that both have been revealed to interact with highly homologous family members of WAVE1 (WAS and WAVE2).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Duke University Medical School, Durham, North Carolina, United States of America.

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.

Show MeSH