Identification of New Intrinsic Proteins in Arabidopsis Plasma Membrane Proteome

Abstract

Identification and characterization of anion channel genes in plants represent a goal for a better understanding of their central role in cell signaling, osmoregulation, nutrition, and metabolism. Though channel activities have been well characterized in plasma membrane by electrophysiology, the corresponding molecular entities are little documented. Indeed, the hydrophobic protein equipment of plant plasma membrane still remains largely unknown, though several proteomic approaches have been reported. To identify new putative transport systems, we developed a new proteomic strategy based on mass spectrometry analyses of a plasma membrane fraction enriched in hydrophobic proteins. We produced from Arabidopsis cell suspensions a highly purified plasma membrane fraction and characterized it in detail by immunological and enzymatic tests. Using complementary methods for the extraction of hydrophobic proteins and mass spectrometry analyses on mono-dimensional gels, about 100 proteins have been identified, 95% of which had never been found in previous proteomic studies. The inventory of the plasma membrane proteome generated by this approach contains numerous plasma membrane integral proteins, one-third displaying at least four transmembrane segments. The plasma membrane localization was confirmed for several proteins, therefore validating such proteomic strategy. 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. This analysis also reveals 10 proteins that display structural properties compatible with transport functions and will constitute interesting targets for further functional studies. Molecular & Cellular Proteomics 3: 675-691, 2004.

Shared Peptides in Mass Spectrometry Based Protein Quantification

“…and AT3G21189(ATPase 9). Earlier reports have suggested that ATPase8,10 are co-expressed evenly over all vegetative tissues, while ATPase 9 is expressed almost exclusively in pollen [9]. In our data, the 3 form a confected component with 6 peptides, and our analysis showed the relative wild-type expression of the 3 to be 34.3%, 57.5%, 8.22% respectively, confirming this observation.…”

Membrane Proteomic Analysis of Arabidopsis thaliana Using Alternative Solubilization Techniques

“…Multiple extractions of purified plasma membranes from Arabidopsis cell suspension cultures with chloroform/ methanol, sodium hydroxide, and Triton X-100 followed by 1-D gel electrophoresis and in-gel tryptic digestion, permitted the identification of 100 plasma membrane proteins by multiple MS analyses. 25 However, only two LRR RLKs were found in that study. Subsequent studies in Arabidopsis using purified plasma membranes from cell suspension cultures incorporated additional enrichment and labeling techniques, which substantially increased the number of LRR RLKs identified.…”

Use of selective extraction and fast chromatographic separation combined with electrophoretic methods for mapping of membrane proteins

“…Using methods, such as differential centrifugation, electrophoresis, or affinity fractionation, cell or tissue lysates are preseparated into fractions containing particular organelles [8,12,14]. After this prefractionation, the "organelle proteome" can be analyzed by use of established proteomics techniques, such as 1-and 2-DE and chromatographic methods followed by MS [8,[12][13][14][15][25][26][27][28].…”

“…After isolation of organelles or subcellular structures, and before electrophoretic separation and final identification by MS, fractionation by selective extraction with different agents and chromatographic separation with different resins can further facilitate proteome analysis [4,12,[23][24][25][26][27][28][29][30].…”