Identification of oxidised proteins in the matrix of rice leaf mitochondria by immunoprecipitation and two-dimensional liquid chromatography-tandem mass spectrometry.

Highly purified mitochondria were isolated from green 7-day-old rice leaves. The mitochondria were sonicated and the matrix fraction isolated as the 100,000g supernatant. Part of the matrix fraction was left untreated while the other part was subjected to a mild oxidative treatment (0.5 mM H2O2+0.2 mM CuSO4 for 10 min at room temperature). The oxidised proteins in both samples were tagged with dinitrophenylhydrazine (DNP), which forms a covalent bond with carbonyl groups. The DNP-tagged proteins were immunoprecipitated using anti-DNP antibodies and digested with trypsin. The mixture of peptides was analysed by nano-HPLC coupled online to an ESI-Quad-TOF mass spectrometer. The peptides were separated by stepwise ion exchange chromatography followed by reverse phase chromatography (2D-LC), and analysed by MS/MS. Proteins were identified by un-interpreted fragment ion database searches. Using this approach we identified 20 oxidised proteins in the control sample and a further 32 in the oxidised sample. Western blots of 2D-gels of the same samples prior to immunoprecipitation verified that the oxidation treatment increases protein oxidation also for specific proteins. Likewise Western blots showed that neither the isolation of mitochondria nor their subfractionation introduced carbonyl groups. We therefore conclude that a number of proteins are oxidised in the matrix of rice leaf mitochondria in vivo and further identify a group of proteins that are particularly susceptible to mild oxidation in vitro.