GAS-PHASE MUDPIT:?LC-FAIMS-MS/MS FOR PROTEOMICS ON AN ION TRAP MASS SPECTROMETER

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The analysis of complex mixtures in proteomics studies is limited by the overall peak capacity of the analysis technique. One way of increasing the peak capacity is by separating peptides prior to the mass spectrometer using long gradients or multidimensional separations. Another way to resolve more peptides is to use an ultra high-resolution mass spectrometer, at a cost of money and instrument complexity (e.g. FT-ICR MS). Here we describe the use of FAIMS (Field Asymmetric Ion Mobility Spectrometry) as a fast gas-phase separation technique interfaced with a bench-top ion trap mass spectrometer for increasing the peak capacity of proteomics analyses without losses in speed or revenue. In FAIMS, ions are separated based on their differential ion mobility in an asymmetric RF field. Ions are then selected by the application of a "compensation voltage" (CV). We have demonstrated the application of FAIMS to proteomics in experiments as follows. The soluble fraction of a whole-cell yeast lysate was digested to peptides using trypsin and analyzed using ?LC and a prototype FAIMS device coupled to an LTQ ion trap mass spectrometer, with preliminary experiments also performed on a homebuilt FAIMS system. We acquired several datasets using a 90 minute reversed phase gradient, with and without FAIMS. For FAIMS-MS experiments, an experimental cycle consisting of one mass-to-charge scan was performed at 8-12 values of CV, spanning the range of useful values of CV. For FAIMS-MS/MS experiments, the cycle consisted of one MS scan followed by two MS/MS scans, at 5 different values of CV. For non-FAIMS experiments, the cycle consisted of one MS scan followed by four MS/MS scans. All data were post-processed using SEQUEST and in-house analysis software.