Improved Sensitivity of Ultralow Flow LC–MS-Based Proteomic Profiling of Limited Samples Using Monolithic Capillary Columns and FAIMS Technology
Michal Greguš, James Kostas, Somak Ray, Susan E. Abbatiello, Alexander R. Ivanov
Abstract
In this work, we pioneered a combination of ultralow flow (ULF) high-efficiency ultranarrow bore monolithic LC columns coupled to MS via a high-field asymmetric waveform ion mobility spectrometry (FAIMS) interface to evaluate the potential applicability for high sensitivity, robust, and reproducible proteomic profiling of low nanogram-level complex biological samples. As a result, ULF LC-FAIMS-MS brought unprecedented sensitivity levels and high reproducibility in bottom-up proteomic profiling. In addition, FAIMS improved the dynamic range, signal-to-noise ratios, and detection limits in ULF LC-MS-based measurements by significantly reducing chemical noise in comparison to the conventional nanoESI interface used with the same ULF LC-MS setup. Two, three, or four compensation voltages separated by at least 15 V were tested within a single LC-MS run using the FAIMS interface. The optimized ULF LC-ESI-FAIMS-MS/MS conditions resulted in identification of 2,348 ± 42 protein groups, 10,062 ± 285 peptide groups, and 15,734 ± 350 peptide-spectrum matches for 1 ng of a HeLa digest, using a 1 h gradient at the flow rate of 12 nL/min, which represents an increase by 38%, 91%, and 131% in respective identifications, as compared to the control experiment (without FAIMS). To evaluate the practical utility of the ULF LC-ESI-FAIMS-MS platform in proteomic profiling of limited samples, approximately 100, 1,000, and 10,000 U937 myeloid leukemia cells were processed, and a one-tenth of each sample was analyzed. Using the optimized conditions, we were able to reliably identify 251 ± 54, 1,135 ± 80, and 2,234 ± 25 protein groups from injected aliquots corresponding to ∼10, 100, and 1,000 processed cells.