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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-captured proteins are MS identifiable.(A) PAX captures a known interacting protein following over-expression in FreeStyle 293 cells. An array of peptides consisting of a proline-rich sequence of FMNL1 and a control sequence were incubated with FreeStyle 293 cell lysate overexpressing srGAP2, photo-crosslinked and washed at high stringency. Immunoblotting with srGAP2 antibody detected phototrapping of srGAP2 by PAX. pBpa in the bait peptides is indicated in blue. (B) Comparative immunoblotting of srGAP2 antigen dot-blot. Different amounts of srGAP2 were spotted onto nitrocellulose membrane and simultaneously immunoblotted with srGAP2 antibody. (C) Identification of srGAP2 by mass spectrometry. The five spots with the highest amount of crosslinked srGAP2 from a duplicate blot of (A) were subjected to MS/MS. 3 unique peptides (underlined) covering 8% of the entire sequence of srGAP2 protein were identified. (D) PAX captures a known interacting protein from mouse brain lysate. Peptide array comprising mGluR5 sequences with a PPXXF motif was incubated with mouse brain lysate, photo-crosslinked and subjected to high stringency washes. Immunoblotting with Homer1 antibody indicates photo-trapping of Homer1 by PAX. pBpa in the bait peptides is indicated in blue.
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pone-0037035-g002: PAX-captured proteins are MS identifiable.(A) PAX captures a known interacting protein following over-expression in FreeStyle 293 cells. An array of peptides consisting of a proline-rich sequence of FMNL1 and a control sequence were incubated with FreeStyle 293 cell lysate overexpressing srGAP2, photo-crosslinked and washed at high stringency. Immunoblotting with srGAP2 antibody detected phototrapping of srGAP2 by PAX. pBpa in the bait peptides is indicated in blue. (B) Comparative immunoblotting of srGAP2 antigen dot-blot. Different amounts of srGAP2 were spotted onto nitrocellulose membrane and simultaneously immunoblotted with srGAP2 antibody. (C) Identification of srGAP2 by mass spectrometry. The five spots with the highest amount of crosslinked srGAP2 from a duplicate blot of (A) were subjected to MS/MS. 3 unique peptides (underlined) covering 8% of the entire sequence of srGAP2 protein were identified. (D) PAX captures a known interacting protein from mouse brain lysate. Peptide array comprising mGluR5 sequences with a PPXXF motif was incubated with mouse brain lysate, photo-crosslinked and subjected to high stringency washes. Immunoblotting with Homer1 antibody indicates photo-trapping of Homer1 by PAX. pBpa in the bait peptides is indicated in blue.

Mentions: We first investigated if proteins photo-trapped to arrayed bait peptides synthesized on cellulose membrane are identifiable by mass spectrometry. The primary concern was whether enough bound protein could be photo-trapped within the peptide spots to be subsequently detected by mass spectrometry. For nanoscale capillary UPLC columns typically used in LC-MS experiments, optimal column loads between 500 ng and 1 ug of total protein digest are desired. Since this direct-interactor only array crosslinking experiment results in a much simplier composition of proteins, column loads as low 100 ng would yield an appropriate column load because the moles of each protein per amount of input material would be significantly higher. As a test case, we ectopically expressed SLIT-ROBO Rho GTPase-activating protein 2 (srGAP2) in FreeStyle 293 suspension cells whose SH3 domain is known to interact with a proline-rich sequence within Formin-like protein 1 (FMNL1) [22]. Cell lysate from transfected cells was incubated with FMNL1 poly-proline peptide arrays with each of the peptide spots containing pBpa at a distinct position (Fig. 2A). The membrane was subjected to UV light (365 nm) to induce cross-linking and then washed extensively with a harsh denaturing solution (0.1% Tween-20, 1% SDS, pH2.5, 95°C: See Method for more details). Immunoblotting was then performed using an anti-srGAP2 antibody, demonstrating that srGAP2 was photo-trapped to the peptide spots on the membrane (Fig. 2A). The amount of the captured srGAP2 varied, dependent on the positions of pBpa within the bait peptide, indicating which amino acids are critical for the interaction. Unexpectedly, the peptide without pBpa also showed positive signal, suggesting this interaction could survive the high stringent washes to a lesser extent. Unrelated peptides did not capture any trace of srGAP2, demonstrating the bound srGAP2 that was detected was specific.


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-captured proteins are MS identifiable.(A) PAX captures a known interacting protein following over-expression in FreeStyle 293 cells. An array of peptides consisting of a proline-rich sequence of FMNL1 and a control sequence were incubated with FreeStyle 293 cell lysate overexpressing srGAP2, photo-crosslinked and washed at high stringency. Immunoblotting with srGAP2 antibody detected phototrapping of srGAP2 by PAX. pBpa in the bait peptides is indicated in blue. (B) Comparative immunoblotting of srGAP2 antigen dot-blot. Different amounts of srGAP2 were spotted onto nitrocellulose membrane and simultaneously immunoblotted with srGAP2 antibody. (C) Identification of srGAP2 by mass spectrometry. The five spots with the highest amount of crosslinked srGAP2 from a duplicate blot of (A) were subjected to MS/MS. 3 unique peptides (underlined) covering 8% of the entire sequence of srGAP2 protein were identified. (D) PAX captures a known interacting protein from mouse brain lysate. Peptide array comprising mGluR5 sequences with a PPXXF motif was incubated with mouse brain lysate, photo-crosslinked and subjected to high stringency washes. Immunoblotting with Homer1 antibody indicates photo-trapping of Homer1 by PAX. pBpa in the bait peptides is indicated in blue.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3351392&req=5

pone-0037035-g002: PAX-captured proteins are MS identifiable.(A) PAX captures a known interacting protein following over-expression in FreeStyle 293 cells. An array of peptides consisting of a proline-rich sequence of FMNL1 and a control sequence were incubated with FreeStyle 293 cell lysate overexpressing srGAP2, photo-crosslinked and washed at high stringency. Immunoblotting with srGAP2 antibody detected phototrapping of srGAP2 by PAX. pBpa in the bait peptides is indicated in blue. (B) Comparative immunoblotting of srGAP2 antigen dot-blot. Different amounts of srGAP2 were spotted onto nitrocellulose membrane and simultaneously immunoblotted with srGAP2 antibody. (C) Identification of srGAP2 by mass spectrometry. The five spots with the highest amount of crosslinked srGAP2 from a duplicate blot of (A) were subjected to MS/MS. 3 unique peptides (underlined) covering 8% of the entire sequence of srGAP2 protein were identified. (D) PAX captures a known interacting protein from mouse brain lysate. Peptide array comprising mGluR5 sequences with a PPXXF motif was incubated with mouse brain lysate, photo-crosslinked and subjected to high stringency washes. Immunoblotting with Homer1 antibody indicates photo-trapping of Homer1 by PAX. pBpa in the bait peptides is indicated in blue.
Mentions: We first investigated if proteins photo-trapped to arrayed bait peptides synthesized on cellulose membrane are identifiable by mass spectrometry. The primary concern was whether enough bound protein could be photo-trapped within the peptide spots to be subsequently detected by mass spectrometry. For nanoscale capillary UPLC columns typically used in LC-MS experiments, optimal column loads between 500 ng and 1 ug of total protein digest are desired. Since this direct-interactor only array crosslinking experiment results in a much simplier composition of proteins, column loads as low 100 ng would yield an appropriate column load because the moles of each protein per amount of input material would be significantly higher. As a test case, we ectopically expressed SLIT-ROBO Rho GTPase-activating protein 2 (srGAP2) in FreeStyle 293 suspension cells whose SH3 domain is known to interact with a proline-rich sequence within Formin-like protein 1 (FMNL1) [22]. Cell lysate from transfected cells was incubated with FMNL1 poly-proline peptide arrays with each of the peptide spots containing pBpa at a distinct position (Fig. 2A). The membrane was subjected to UV light (365 nm) to induce cross-linking and then washed extensively with a harsh denaturing solution (0.1% Tween-20, 1% SDS, pH2.5, 95°C: See Method for more details). Immunoblotting was then performed using an anti-srGAP2 antibody, demonstrating that srGAP2 was photo-trapped to the peptide spots on the membrane (Fig. 2A). The amount of the captured srGAP2 varied, dependent on the positions of pBpa within the bait peptide, indicating which amino acids are critical for the interaction. Unexpectedly, the peptide without pBpa also showed positive signal, suggesting this interaction could survive the high stringent washes to a lesser extent. Unrelated peptides did not capture any trace of srGAP2, demonstrating the bound srGAP2 that was detected was specific.

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
Related in: MedlinePlus