Large-scale proteome analysis of tomato fruit microsomes

Solanum lycopersicum (tomato) is one of the most important plant crops and a model system to study development and ripening of fleshy fruit. The membrane system is very important for all biological processes; for example, the endoplasmic reticulum (ER) and the Golgi apparatus play a pivotal role in the secretion of proteins and in the synthesis of the non-cellulosic portion of the cell wall. The aim of this work was to characterize the membrane proteome profile of tomato fruit at the ‘‘mature green’’ ripening stage. Total microsomes were prepared and separated by centrifugation through a iodixanol continuous gradient. The organelle distribution pattern (plasma membrane, ER, Golgi, chloroplast, nucleus) was determined using known markers of these compartments. Proteins were identified using a combination of 1-D SDS polyacrylamide gel electrophoresis and nanoLC-ESIMS/ MS. After electrophoresis, each lane was cut into 14 pieces, digested with trypsin and analyzed by nano-HPLC coupled to an Orbitrap mass spectrometer. More than 2400 different proteins were identified, for which the Arabidopsis homologue was searched and subjected to GO analysis to determine the represented biological process, molecular function and subcellular localization. GO term enrichment analysis confirmed the enrichment in membrane proteins. This large scale proteomic analysis provides a detailed reference map of the membrane proteome at specific stages of tomato fruit development and a background for comparison of physiological processes such as ripening or biotic and abiotic stresses.