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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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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			Action	DRS
	F027014_rep1.dat	Other
	F027015_rep2.dat	Other
	F027016_rep3.dat	Other
	replicate1.zip	Other
	replicate2.zip	Other