Proteomic analysis of highly purified peroxisomes from etiolated soybean cotyledons

To identify previously unknown peroxisomal proteins, we established an optimized method for isolating highly purified peroxisomes from etiolated soybean cotyledons using Percoll density gradient centrifugation followed by iodixanol density gradient centrifugation. Proteins in highly purified peroxisomes were separated by two-dimensional PAGE. We performed peptide mass fingerprinting of proteins separated in the gel with matrix-assisted laser desorption ionization time-of-flight mass spectrometry and used the peptide mass fingerprints to search a non-redundant soybean expressed sequence tag database. We succeeded in assigning 92 proteins to 70 sequences in the database. Among them, proteins encoded by 30 sequences were judged to be located in peroxisomes. These included enzymes for fatty acid -oxidation, the glyoxylate cycle, photorespiratory glycolate metabolism, stress response and metabolite transport. We also show experimental evidence that plant peroxisomes contain a short-chain dehydrogenase/reductase family protein, enoyl-CoA hydratase/isomerase family protein, 3-hydroxyacyl-CoA dehydrogenase-like protein and a voltage-dependent anion-selective channel protein.