title: Improved two-dimensional reversed phase-reversed phase LC-MS/MS approach for identification of peptide-protein interactions.
abstract: Quantitative mass spectrometry (MS) in combination with affinity purification approaches allows for an unbiased study of protein-protein and peptide-protein interactions. In shotgun approaches that are based on proteolytic digestion of complex protein mixtures followed by two-dimensional liquid-phase chromatography, the separation effort prior to MS analysis is focused on tryptic peptides. Here we developed an improved offline 2-D liquid chromatography-MS/MS approach for the identification and quantification of binding proteins utilizing reversed-phase capillary columns with acidic acetonitrile-containing eluents in both chromatographic dimensions. A specific fractionation scheme was applied in order to obtain samples with evenly distributed peptides and to fully utilize the separation space in the second dimension nanoLC-MS/MS. We report peptide-protein interaction studies to identify phosphorylation-dependent binding partners of the T cell adapter protein ADAP. The results of the SILAC-based pull-down experiments show this approach is well suited for distinguishing phosphorylation-specific interactions from unspecific binding events. The data provide further evidence that phosphorylated Tyr 595 of ADAP may serve as a direct binding site for the SH2 domains of the T cell proteins SLP76 and NCK. From a technical point of view we provide a detailed protocol for an offline 2-D RP-RP LC-MS/MS method that offers a robust and time-saving alternative for quantitative interactome analysis.
authors.affiliation: Leibniz-Institut für Molekulare Pharmakologie, Robert-Rössle-Str. 10, 13125 Berlin, Germany.
authors.forename: Heike
authors.initials: H
authors.lastname: Stephanowitz
authors.forename: Sabine
authors.initials: S
authors.lastname: Lange
authors.forename: Diana
authors.initials: D
authors.lastname: Lang
authors.forename: Christian
authors.initials: C
authors.lastname: Freund
authors.forename: Eberhard
authors.initials: E
authors.lastname: Krause
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry / Q000378:metabolism; D056148:Chromatography, Reverse-Phase / Q000379:methods*; D006801:Humans; D007553:Isotope Labeling; D019169:Jurkat Cells; D010446:Peptide Fragments / Q000737:chemistry* / Q000378:metabolism; D010766:Phosphorylation; D025941:Protein Interaction Mapping / Q000379:methods*; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D015203:Reproducibility of Results; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D048868:Adaptor Proteins, Signal Transducing; C106957:FYB1 protein, human; D010446:Peptide Fragments; D011506:Proteins