(215e) Integrated Top-Down And Bottom-Up Strategy For Characterizing Intact Proteins And Their Modifications

Two complementary strategies for protein characterization are typically employed using mass spectrometry: bottom-up and top-down, each with its own strengths and weaknesses. We present an integrated top-down / bottom-up approach facilitated by concurrent LC/MS analysis and fraction collection. For comprehensive high-throughput intact protein profiling, we employed reversed phase LC separations coupled on-line with a 12T Fourier transform ion cyclotron resonance (FTICR) mass spectrometry. Confident assignments of intact protein masses and their gas phase fragment spectra are enabled by limiting the bottom-up analysis to individual fractions and thereby reducing the complexity of the protein mixture undergoing analysis. For large-scale proteome profiling, additional dimension of protein separation technique, such as free flow electrophoresis (FFE), might be employed to reduce the complexity and improve the quality of MS. Herein, we apply this novel comprehensive strategy to elucidate various biological phenomena such as dynamics of protein modifications under conditions of oxidative stress.