Proteome profiling during oxidative stress. Scheme delineating the experimental procedures applied to measure protein levels at different time points immediately before and after exposure to H2O2. Cells were harvested at the indicated time points, followed by preparation of protein lysates and digestion of proteins into peptides. Peptides were separated by strong ion exchange SCX chromatography into 24 fractions. Each fraction was separated by reversed phase chromatography and directly eluted into a ThermoFisher LTQ Orbitrap mass spectrometer (for details see Materials and methods). Transcript levels were determined from the same samples using DNA microarrays. Proteins and mRNAs were measured in two independent biological repeats of the time-course experiment, and quantified using QTools . MS/MS, tandem mass spectrometry.