Qualitative and Quantitative Characterization of Proteomes Using Ion Mobility Mass Spectrometry with Data Independent Acquisition.

A central goal in a proteomics study is to fully characterize a sample both qualitatively and quantitatively. A data-independent analysis yields reproducible fragmentation and peak area information for peptides in a complex mixture (1). The addition of ion mobility into this analysis inserts an orthogonal, gas-phase separation, occurring in the millisecond timescale, which is poised nicely between chromatographic and TOF mass spectrometry timescales. Peptides and their corresponding fragment ions are aligned by chromatographic retention time in dataindependent analyses. When ion mobility is used, peptides and their fragment ions also share the same mobility drift time which dramatically improves specificity. In this study, eukaryotic and prokaryotic samples were analyzed by one-dimensional (1D) and 2D chromatography using high-low pH RP-RP separations, both with and without ion mobility separation. The number of proteins identified in a sample doubled by using a five fraction 2D separation compared to 1D chromatography. Utilizing the mobility separation yielded an additional increase in the number of proteins and peptides by at least 20%, without any additional instrument time. The amount of information obtained from a sample depended on the amount loaded as well as the time dedicated to analysis of the sample. For one prokaryotic sample, 552 proteins were reproducibly identified with 1D chromatography without ion mobility from 0.75 ug of material. Loading 5 ug and performing 5 fractions, along with ion mobility, yielded 1260 reproducible protein identifications. Stoichiometry between proteins was determined by comparing the average intensity of the top three peptides to every protein to that of an internal standard (2). Agreement was found between the experimental stoichiometric ratios and those found in literature for many protein groups. 1. Geromanos, S.J., et. al. Proteomics. 2009 (6):1683–95. 2. Silva, J.C., et. al. Mol Cell Proteomics. 2006 (1):144–56.