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. 2011 Sep 2;10(9):4158-64.
doi: 10.1021/pr200578n. Epub 2011 Aug 2.

Proteomic Screening Method for Phosphopeptide Motif Binding Proteins Using Peptide Libraries

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Free PMC article

Proteomic Screening Method for Phosphopeptide Motif Binding Proteins Using Peptide Libraries

Heather R Christofk et al. J Proteome Res. .
Free PMC article

Abstract

Phosphopeptide binding domains mediate the directed and localized assembly of protein complexes essential to intracellular kinase signaling. To identify phosphopeptide binding proteins, we developed a proteomic screening method using immobilized partially degenerate phosphopeptide mixtures combined with SILAC and microcapillary LC-MS/MS. The method was used to identify proteins that specifically bound to phosphorylated peptide library affinity matrices, including pTyr, and the motifs pSer/pThr-Pro, pSer/pThr-X-X-X-pSer/pThr, pSer/pThr-Glu/Asp, or pSer/pThr-pSer/pThr in degenerate sequence contexts. Heavy and light SILAC lysates were applied to columns containing these phosphorylated and nonphosphorylated (control) peptide libraries respectively, and bound proteins were eluted, combined, digested, and analyzed by LC-MS/MS using a hybrid quadrupole-TOF mass spectrometer. Heavy/light peptide ion ratios were calculated, and peptides that yielded ratios greater than ∼3:1 were considered as being from potential phosphopeptide binding proteins since this ratio represents the lowest ratio from a known positive control. Many of those identified were known phosphopeptide-binding proteins, including the SH2 domain containing p85 subunit of PI3K bound to pTyr, 14-3-3 bound to pSer/pThr-Asp/Glu, polo-box domain containing PLK1 and Pin1 bound to pSer/pThr-Pro, and pyruvate kinase M2 binding to pTyr. Approximately half of the hits identified by the peptide library screens were novel. Protein domain enrichment analysis revealed that most pTyr hits contain SH2 domains, as expected, and to a lesser extent SH3, C1, STAT, Tyr phosphatase, Pkinase, C2, and PH domains; however, pSer/pThr motifs did not reveal enriched domains across hits.

Figures

Figure 1
Figure 1
A proteomic screen to identify phosphopeptide binding proteins from cell lysates. Biotinylated partially degenerate peptide libraries were incubated with streptavidin beads and packed onto columns to form the phosphorylated and non-phosphorylated peptide library affinity matrices. Heavy SILAC lysates were flowed over the phosphorylated peptide library column while light SILAC lysates were flowed over the non-phosphorylated peptide library column. Bound proteins were eluted with 20 mM sodium phenylphosphate (in the pTyr peptide library screen) or free peptide library (in the pSer/pThr peptide library screens), digested with trypsin, and analyzed by LC/MS/MS. Proteins were identified and heavy:light ratios were calculated using commercially available automated software. An example spectrum from the pTyr phosphopeptide binding screen showing that p85 specifically binds to the pTyr peptide library.
Figure 2
Figure 2
Results from the four screens for phosphopeptide binding proteins yielding greater than ~3:1 heavy:light SILAC ratios. Each protein identified by LC/MS/MS is represented as a black diamond. The names of known (previously published) phosphopeptide binding proteins are shown in blue, the names of candidate novel phosphopeptide binding proteins are shown in red and the phosphopeptide binding proteins across multiple libraries are shown in black. (A). Screen results for the pTyr peptide library. (B). Screen results for the pSer/pThr-Pro peptide library. (C). Screen results for the pSer/pThr-X-X-X-pSer/pThr peptide library. (D). Screen results for the pSer/pThr-Asp/Glu peptide library.
Figure 2
Figure 2
Results from the four screens for phosphopeptide binding proteins yielding greater than ~3:1 heavy:light SILAC ratios. Each protein identified by LC/MS/MS is represented as a black diamond. The names of known (previously published) phosphopeptide binding proteins are shown in blue, the names of candidate novel phosphopeptide binding proteins are shown in red and the phosphopeptide binding proteins across multiple libraries are shown in black. (A). Screen results for the pTyr peptide library. (B). Screen results for the pSer/pThr-Pro peptide library. (C). Screen results for the pSer/pThr-X-X-X-pSer/pThr peptide library. (D). Screen results for the pSer/pThr-Asp/Glu peptide library.
Figure 3
Figure 3
(A). The Pfam protein domain enrichment results from the hits of the pTyr phosphopeptide binding screen showing an enrichment of SH2 domains. (B). The String predicted protein-protein interaction (PPI) network among the pTyr screen hits.

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