14-3-3 membrane protein interactome

To elucidate more details of the interaction between the PM (plasma membrane) H+ ATPase and 14-3-3 and to identify novel interaction partners of this multi-protein complex we developed a strategy for identification of protein-protein interactions using /in vivo/ formaldehyde cross-linking in combination with immunoprecipitation and MS-based protein identification. This method identified numerous proteins which may interact with the PM H+ ATPase to regulate its activity or to transmit environmental signals across the plasma membrane. All tryptic peptides mixtures were analyzed by LC-MS/MS using a nanoflow Easy-nLC System (Thermo Scientific, http://www.thermo.com) and an LTQ-Orbitrap hybrid mass spectrometer(Thermo Scientific) as a mass analyser. Peptides were separated on a 75 µm C18 analytical column (Integrafrit Biobasic, New Objectives, USA) with a linear gradient (10 to 64% acetonitrile) and sprayed directly into the LTQ-Orbitrap mass spectrometer. Data processing: Fragmentation spectra were searched against the NCBI non-redundant protein database (www.ncbi.nlm.nih.gov) with taxonomic restriction to all plant species using the Mascot software (version 2.2, Matrix Science, UK; www.matrixscience.com ) with the following search parameters: trypsin as cleavage enzyme, peptide mass tolerance 300 ppm, MS/MS tolerance 0.8 Da, cysteine carbamidomethylation as fixed modification, methionine oxidation as variable modification. Only peptides longer than 5 amino acids were considered. Peptides were accepted automatically if they displayed a Mascot score higher than 47 which was the Mascot significance score threshold (p < 0.01). False positive rate as estimated from a search against the decoy NCBI database was 1.34%.