Arabidopsis leaf plasma membrane proteome using a gel free method: Focus on receptor–like kinases

The hydrophobic proteins of plant plasma membrane still remain largely unknown. For example in the Arabidopsis genome, receptor-like kinases (RLKs) are plasma membrane proteins, functioning as the primary receptors in the signaling of stress conditions, hormones and the presence of pathogens form a diverse family of over 610 genes. A limited number of these proteins have appeard in protein profiles. The detection of these proteins and thus the determination of their dynamics and tissue specificity, is technically challenging due to their low abundance and association to a lipid membrane. To identify new putative membrane proteins especially receptor systems, we used a gel free proteomic strategy based on mass spectrometry analyses of a plasma membrane fraction enriched in hydrophobic proteins. We produced from Arabidopsis leaf a highly purified plasma membrane fraction with the aqueous two-phase partitioning technique. By separating the proteins in the plasma membrane fraction with ion exchange and reverse phase chromatography and analyzing the resulting fractions on a MALDI-TOF mass spectrometer, over 900 proteins were detected. The plasma membrane proteome generated by this approach contains numerous plasma membrane integral proteins, onethird displaying at least four trans-membrane segments. An in silico analysis shows a correlation between the putative functions of the identified proteins and the expected roles for plasma membrane in transport, signaling, cellular traffic and metabolism. Of these proteins, 304 were annotated as membrane proteins, 69 were RLKs, distributed among the different receptor families in proportions reflecting the distribution in the genome. Of the RLKs that were identified, most are reported for the first time at the protein level and will constitute interesting targets for further functional studies.