Dataset Information

14-3-3 membrane protein interactome

ABSTRACT: 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%.

INSTRUMENT(S): LTQ Orbitrap

ORGANISM(S): Lilium

TISSUE(S): Pollen Tube

SUBMITTER: Waltraud Schulze

LAB HEAD: Waltraud Schulze

PROVIDER: PXD000244 | Pride | 2016-05-10

REPOSITORIES: Pride

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Publications

In vivo cross-linking combined with mass spectrometry analysis reveals receptor-like kinases and Ca(2+) signalling proteins as putative interaction partners of pollen plasma membrane H(+) ATPases.

Pertl-Obermeyer Heidi H Schulze Waltraud X WX Obermeyer Gerhard G

Journal of proteomics 20140511

During fertilisation in plants, pollen grains germinate and generate a pollen tube which grows through the style tissue to the egg apparatus delivering the two sperm cells for fertilisation. For this process, adaption to specific environmental conditions and communication between male and female organs are essential, requiring the sensing of internal and external signals which are translated into tube growth. The plasma membrane (PM) H(+) ATPase energises the pollen plasma membrane for nutrient, ...[more]

PMID: 24824344

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Project description:Proteomic response of adult Drosophila melanogaster acclimated at three contrasted thermal conditions (11, 25 and 31 degree Celsius) investigated by 2D-DIGE experiment. Thermal acclimation drastically alters thermotolerance of ectotherms, but the mechanisms determining this plastic response are not fully understood. The present study investigates the proteomic response (2D-DIGE) of adult Drosophila melanogaster acclimated at 11, 25 or 31 °C. As expected 11 °C-acclimation improved cold tolerance and 31 °C-acclimation improved heat tolerance. We hypothesized that the marked organismal responses to acclimation could be detected at the proteomic level assuming that changes in the abundance of specific proteins are linked to the physiological changes underlying the phenotypic response. The 31 °C-acclimated flies displayed a particular divergent proteomic profile where molecular chaperones made up a large number of the proteins that were modulated during heat acclimation. Many other proteins showed significant modulation during acclimation including proteins involved in iron ion and cell redox homeostasis, carbohydrate and energy metabolism, chromatin remodeling and translation, and contractile machinery. Interestingly the changes in protein abundance were often unrelated to transcriptional activity of the genes coding for the proteins, except for the most strongly expressed proteins (e.g. Hsp70). The 11 °C-acclimation evoked weak proteomic response despite the marked effect on the organismal phenotype. Thus the acquired cold tolerance observed here may involve regulatory process such as posttranslational regulation rather than de novo protein synthesis. Bioinformatics and data processing: The proteinScape 2.1 software (BrukerDaltonik GmbH) was used to submit MS/MS data to the following database: NCBI restricted to Drosophila (June 2011, 223,543 sequences) using the Mascot search engine (Mascot server v2.2, http://www.matrixscience.com). Parameters were set as follows: trypsin as enzyme with one allowed miscleavage, carbamidomethylation of cysteins as fixed modification and methionine oxidation as variable modifications. The mass tolerance for parent and fragment ions was set to 0.5 Da. Peptide identifications were accepted if the individual ion Mascot scores were above the identity threshold (the ion score is −10*log(P), where P is the probability that the observed match is a random event, P < 0.05). In case of ambiguous assignments (one compound fit to more than one peptide), peptide were accepted based on the peptide score, meaning that the peptide sequence with the highest score is accepted. The compilation of identified peptides to proteins was performed with the ProteinExtractor algorithm (Thiele et al., 2008, 2010), so that every protein reported was identified by at least one peptide with significant ion Mascot score (above the identity threshold).

Dataset Information

14-3-3 membrane protein interactome

Publications

In vivo cross-linking combined with mass spectrometry analysis reveals receptor-like kinases and Ca(2+) signalling proteins as putative interaction partners of pollen plasma membrane H(+) ATPases.

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