Proteomics of the chloroplast: Systematic identification and targeting analysis of lumenal and peripheral thylakoid proteins

The soluble and peripheral proteins in the thylakoids of pea were systemati cally analyzed by using two-dimensional electrophoresis, mass spectrometry, and N-terminal Edman sequencing, followed by database searching. After cor recting to eliminate possible isoforms and post-translational modifications , we estimated that there are at least 200 to 230 different lumenal and per ipheral proteins. Sixty-one proteins were identified; for 33 of these prote ins, a clear function or functional domain could be identified, whereas for 10 proteins, no function could be assigned. For 18 proteins, no expressed sequence tag or full-length gene could be identified in the databases, desp ite experimental determination of a significant amount of amino acid sequen ce. Nine previously unidentified proteins with lumenal transit peptides are presented along with their full-length genes; seven of these proteins poss ess the twin arginine motif that is characteristic for substrates of the TA T pathway. Logoplots were used to provide a detailed analysis of the lumena l targeting signals, and all nuclear-encoded proteins identified on the two -dimensional gels were used to test predictions for chloroplast localizatio n and transit peptides made by the software programs ChloroP, PSORT, and Si gnalP, A combination of these three programs was found to provide a useful tool for evaluating chloroplast localization and transit peptides and also could reveal possible alternative processing sites and dual targeting. The potential of proteomics for plant biology and homology-based searching with mass spectrometry data is discussed.