Mass spectroscopy and activity analysis of oxidized peroxisomal proteins

In our previous work peroxisomal catalase (CAT) and malate dehydrogenase activity were shown to be inhibited by oxidizing conditions. Our objective was to investigate peroxisomal protein oxidization as a source of this inhibition. To accomplish this persoxisomal samples were harvested from castor beans in the presence or absence of 100 mM H 2 O 2 . Half of the samples were isolated in the presence of dithiothreitol (DTT) and subjected to further oxidation by incubation with Cu (II) and ascorbate (Cu/Asc) or H 2 O 2 . Oxidized proteins were detected by 2,4‐dinitrophenylhydrazine derivatization followed by anti‐DNP antibody immunoblotting. Our results revealed several proteins were oxidized when peroxisomes were isolated in the absence of DTT and/or treated with Cu/Asc or H 2 O 2 . Individual proteins separated by gel electrophoresis were digested with trypsin and analyzed with MALDI‐TOF mass spectroscopy, confirming the identity of catalase (CAT) and isocitrate lyase (ICL). Mass spectroscopy also revealed the presence of an unknown oxidized peptide. Expasy PeptideMass computational analysis along with custom comparative software was used to locate the oxidized peptide within the amino acid (AA) sequence. The location was found within AA 234–242 in CAT 2. The CAT 2 protein was then mapped using Cn3D software and the oxidized peptide was found to be located near the active site. This data may explain our previous findings.