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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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Experimental outline of PAX methodology.(A) Arrays of bait peptides are synthesized onto PEG-based membrane supports. The photo-activatable amino acid cross-linker (pBpa) is incorporated into the bait peptides so that each peptide spot has pBpa at a different position in the sequence (see inset schematic). (B, C) The membrane is incubated with cell lysate and subjected to 350–365 nm light to cross-link with interacting proteins. (D) The indirect and non-specific interactors are removed by high stringent, denaturing washes. (E, F) Each strip of the bait peptide spots is cut off the membrane, further chopped into small pieces, and trypsinized. (G, H) The samples are filtered to remove PEG and subjected to LC-MS/MS analysis to identify the photo-trapped proteins.
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pone-0037035-g001: Experimental outline of PAX methodology.(A) Arrays of bait peptides are synthesized onto PEG-based membrane supports. The photo-activatable amino acid cross-linker (pBpa) is incorporated into the bait peptides so that each peptide spot has pBpa at a different position in the sequence (see inset schematic). (B, C) The membrane is incubated with cell lysate and subjected to 350–365 nm light to cross-link with interacting proteins. (D) The indirect and non-specific interactors are removed by high stringent, denaturing washes. (E, F) Each strip of the bait peptide spots is cut off the membrane, further chopped into small pieces, and trypsinized. (G, H) The samples are filtered to remove PEG and subjected to LC-MS/MS analysis to identify the photo-trapped proteins.

Mentions: Benzophenone photoprobes, which covalently couple to unreactive C-H bonds upon exposure to 350–360 nm light, have been widely used for biochemical characterization of macromolecules since the 1980s [13], [14]. More recently the benzophenone phenylalanine derivative, p-benzoyl-L-phenylalanine (pBpa) has been successfully translated into proteins of interest within cells, enabling the photo-induced crosslinking of protein-protein interactions as they occur in situ [15], [16], [17], [18], [19]. We have used this approach to purify and discover new interactions by incorporating pBpa into protein binding domains expressed within mammalian cells [20]. The success of this approach prompted us to wonder if the reverse strategy, utilizing peptides containing pBpa, could also be useful for the rapid identification of protein interactions from cell lysates. The primary obstacle to this approach is that pBpa must be positionally placed within the peptide such that it is close enough to capture a binding protein, but not within the binding site so as to not disrupt the relevant interaction. This requirement creates a problem, however, since permissive positions within a putative peptide ligand are unknown for uncharacterized interactions that are the target of protein interaction discovery approaches. This problem could be circumvented, however, if the peptide ligand is represented as an array of peptide sequences, with pBpa placed at each possible position within the peptide (Figure 1A and inset). In this format, potential interactions with the peptide may be covalently captured by those peptides in which pBpa is at a permissive position for binding and crosslinking, without prior knowledge of which site is optimal. Previously we have used the SPOT technology to synthesize arrays of peptide sequences on PEG derivatized cellulose membranes [20], [21], [22]. 384 peptides can be synthesized on a membrane within a 24×16 spot format, and up to 4 membranes can be processed at a time. We hypothesized that pBpa containing peptides arrayed in this format, incubated with cell or tissue lysates, and exposed to UV light in the 350–360 nm range might provide a convenient and rapid approach to phototrap peptide-protein interactions. After incubation with cell/tissue lysates of interest and photo-induced covalent trapping of binding proteins (Figure 1B–C), the peptide array can be extensively washed under denaturing conditions to aid the removal of indirect and non-specific interactors (Figure 1D). Peptides with bound proteins would then be trypsin digested and analyzed by mass spectrometry (Figure 1E–H).


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)

Experimental outline of PAX methodology.(A) Arrays of bait peptides are synthesized onto PEG-based membrane supports. The photo-activatable amino acid cross-linker (pBpa) is incorporated into the bait peptides so that each peptide spot has pBpa at a different position in the sequence (see inset schematic). (B, C) The membrane is incubated with cell lysate and subjected to 350–365 nm light to cross-link with interacting proteins. (D) The indirect and non-specific interactors are removed by high stringent, denaturing washes. (E, F) Each strip of the bait peptide spots is cut off the membrane, further chopped into small pieces, and trypsinized. (G, H) The samples are filtered to remove PEG and subjected to LC-MS/MS analysis to identify the photo-trapped proteins.
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Related In: Results  -  Collection

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pone-0037035-g001: Experimental outline of PAX methodology.(A) Arrays of bait peptides are synthesized onto PEG-based membrane supports. The photo-activatable amino acid cross-linker (pBpa) is incorporated into the bait peptides so that each peptide spot has pBpa at a different position in the sequence (see inset schematic). (B, C) The membrane is incubated with cell lysate and subjected to 350–365 nm light to cross-link with interacting proteins. (D) The indirect and non-specific interactors are removed by high stringent, denaturing washes. (E, F) Each strip of the bait peptide spots is cut off the membrane, further chopped into small pieces, and trypsinized. (G, H) The samples are filtered to remove PEG and subjected to LC-MS/MS analysis to identify the photo-trapped proteins.
Mentions: Benzophenone photoprobes, which covalently couple to unreactive C-H bonds upon exposure to 350–360 nm light, have been widely used for biochemical characterization of macromolecules since the 1980s [13], [14]. More recently the benzophenone phenylalanine derivative, p-benzoyl-L-phenylalanine (pBpa) has been successfully translated into proteins of interest within cells, enabling the photo-induced crosslinking of protein-protein interactions as they occur in situ [15], [16], [17], [18], [19]. We have used this approach to purify and discover new interactions by incorporating pBpa into protein binding domains expressed within mammalian cells [20]. The success of this approach prompted us to wonder if the reverse strategy, utilizing peptides containing pBpa, could also be useful for the rapid identification of protein interactions from cell lysates. The primary obstacle to this approach is that pBpa must be positionally placed within the peptide such that it is close enough to capture a binding protein, but not within the binding site so as to not disrupt the relevant interaction. This requirement creates a problem, however, since permissive positions within a putative peptide ligand are unknown for uncharacterized interactions that are the target of protein interaction discovery approaches. This problem could be circumvented, however, if the peptide ligand is represented as an array of peptide sequences, with pBpa placed at each possible position within the peptide (Figure 1A and inset). In this format, potential interactions with the peptide may be covalently captured by those peptides in which pBpa is at a permissive position for binding and crosslinking, without prior knowledge of which site is optimal. Previously we have used the SPOT technology to synthesize arrays of peptide sequences on PEG derivatized cellulose membranes [20], [21], [22]. 384 peptides can be synthesized on a membrane within a 24×16 spot format, and up to 4 membranes can be processed at a time. We hypothesized that pBpa containing peptides arrayed in this format, incubated with cell or tissue lysates, and exposed to UV light in the 350–360 nm range might provide a convenient and rapid approach to phototrap peptide-protein interactions. After incubation with cell/tissue lysates of interest and photo-induced covalent trapping of binding proteins (Figure 1B–C), the peptide array can be extensively washed under denaturing conditions to aid the removal of indirect and non-specific interactors (Figure 1D). Peptides with bound proteins would then be trypsin digested and analyzed by mass spectrometry (Figure 1E–H).

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