Identification of carbonylated proteins by MALDI-TOF mass spectroscopy reveals susceptibility of ER.

Reactive oxygen species are produced by metabolism over time, but can also be produced in more acute conditions of cell stress such as treatment with cytotoxic drugs. Treatment of HL-60 cells with peroxide results in cell death and protein carbonylation, a non-enzymatic protein modification that typically results from oxidative stress within cells. It has recently become clear that protein carbonylation during ageing is confined to specific proteins. It is therefore of interest to be able to identify which proteins are susceptible to protein carbonylation. Here we demonstrate immunoprecipitation of carbonylated proteins coupled with 2D-gel electrophoresis to identify carbonylated proteins by MALDI-TOF m/s fingerprinting. The results show that some ER proteins are readily carbonylated in response to peroxide treatment of HL-60 cells. This is likely to have implications for the induction of cell death in such cells.