Quantitative measurement of specific biomarkers for protein oxidation, nitration and glycation in Arabidopsis leaves.

Higher plants are continually exposed to reactive oxygen and nitrogen species during their lives. Together with glucose and reactive dicarbonyls, these can modify proteins spontaneously, leading to protein oxidation, nitration and glycation. These reactions have the potential to damage proteins and have an impact on physiological processes. The levels of protein oxidation, nitration and glycation adducts were assayed, using liquid chromatography coupled with tandem mass spectrometry, in total leaf extracts over a diurnal cycle and when exposed to conditions that promote oxidative stress. Changes in the levels of oxidation, glycation and nitration adducts were found between the light and dark phases under non-stress conditions. A comparison between wild-type plants and a mutant lacking peptide methionine sulfoxide reductase (pmsr2-1) showed increased protein oxidation, nitration and glycation of specific amino acid residues during darkness in pmsr2-1. Short-term excess light exposure, which promoted oxidative stress, led to increased protein glycation, specifically by glyoxal. This suggested that any increased oxidative damage to proteins was within the repair capacity of the plant. The methods developed here provide the means to simultaneously detect a range of protein oxidation, nitration and glycation adducts within a single sample. Thus, these methods identify a range of biomarkers to monitor a number of distinct biochemical processes that have an impact on the proteome and therefore the physiological state of the plant.