Targeted Analysis of Protein Phosphorylation by 2D Electrophoresis.

Two-dimensional (2D) gel electrophoresis combines isoelectric focusing in the first and SDS polyacrylamide gel electrophoresis in the second dimension to separate complex mixtures of proteins with unequalled resolution and sensitivity. It is well suited for the analysis of posttranslational protein modifications as most of them affect the isoelectric point and, therefore, the focusing behavior of the protein in the first dimension. It is particularly useful for low-abundance proteins, as it provides a first indication of PTMs, before establishing methods for protein isolation. For targeted proteomics of more abundant proteins, 2D electrophoresis itself may be the method of choice for the isolation of posttranslationally modified isoforms of the protein of interest for mass spectrometric analyses. Protein phosphorylation can be detected by use of phospho-specific stains or antibodies, or by comparing spot patterns of a protein sample before and after phosphatase treatment. Here we describe a simple method, combining 2D gel electrophoresis and western blot analysis with dephosphorylation by λ-phosphatase to analyze the phosphorylation status of oxophytodienoic acid reductase 3 in protein extracts from different organs of tomato and Arabidopsis plants.

Rhomboid proteins in the chloroplast envelope affect the level of allene oxide synthase in Arabidopsis thaliana.

Rhomboids are intra-membrane serine proteases whose sequences are found in nearly all organisms. They are involved in a variety of biological functions in both eukaryotes and prokaryotes. Localization assays revealed that two Arabidopsis thaliana rhomboid-like proteases (AtRBL), AtRBL8 and AtRBL9, are targeted to the chloroplast. Using transgenic plants expressing epitope-tagged AtRBL9, we localized AtRBL9 to the chloroplast inner envelope membrane, with both its N- and C-termini facing the stroma. Mass spectrometry analyses confirmed this localization, and suggested that this is also the case for AtRBL8. Both are proteins of very low abundance. The results of size-exclusion chromatography implied that AtRBL9 forms homo-oligomers. In search of a putative function, a comparative proteomic analysis was performed on wild-type and double-knockout plants, lacking both AtRBL8 and AtRBL9, using the iTRAQ method. Of 180 envelope proteins, the level of only a few was either increased or decreased in the mutant line. One of the latter, allene oxide synthase, is involved in jasmonic acid biosynthesis. This observation provides an explanation for the recently reported aberration in flower morphology that is associated with the loss of AtRBL8.