Coupling High-Field Asymmetric Ion Mobility Spectrometry with Capillary Electrophoresis-Electrospray Ionization-Tandem Mass Spectrometry Improves Protein Identifications in Bottom-Up Proteomic Analysis of Low Nanogram Samples.

In this work, we pioneered the assessment of coupling high-field asymmetric waveform ion mobility spectrometry (FAIMS) with ultrasensitive capillary electrophoresis hyphenated with tandem mass spectrometry (CE-MS/MS) to achieve deeper proteome coverage of low nanogram amounts of digested cell lysates. An internal stepping strategy using three or four compensation voltages per analytical run with varied cycle times was tested to determine optimal FAIMS settings and MS parameters for the CE-FAIMS-MS/MS method. The optimized method applied to bottom-up proteomic analysis of 1 ng of HeLa protein digest standard identified 1314 ± 30 proteins, 4829 ± 200 peptide groups, and 7577 ± 163 peptide spectrum matches (PSMs) corresponding to a 16, 25, and 22% increase, respectively, over CE-MS/MS alone, without FAIMS. Furthermore, the percentage of acquired MS/MS spectra that resulted in PSMs increased nearly 2-fold with CE-FAIMS-MS/MS. Label-free quantitation of proteins and peptides was also assessed to determine the precision of replicate analyses from FAIMS methods with increased cycle times. Our results also identified from 1 ng of HeLa protein digest without any prior enrichment 76 ± 9 phosphopeptides, 18% of which were multiphosphorylated. These results represent a 46% increase in phosphopeptide identifications over the control experiments without FAIMS yielding 2.5-fold more multiphosphorylated peptides.