Characterization of Phosphorylated Peptides Using Traveling Wave-Based and Drift Cell Ion Mobility Mass Spectrometry

Phosphorylation is one the most studied and important post translational modifications. Nano electrospray mass spectrometry coupled with traveling wave (T-Wave)-based ion mobility has been used to filter for phosphorylated peptides in tryptic protein digests. T-Wave parameters have been optimized to maximize the separation between phosphorylated and non-phosphorylated peptides. A method to calibrate the T-Wave device, to provide estimates of collision cross sections, is presented, and these estimates are in excellent agreement with values obtained on drift cell instrumentation. Phosphorylated peptides have smaller cross sections which enables their separation from non-phosphorylated peptides of the same m/z. Post-mobility fragmentation is used to obtain the primary sequence for peptides of interest. This approach is shown to have potential as an additional screen for phosphorylated peptides, where up to 40% of observed peptides can be eliminated from the study.