Improved Sensitivity of Ultralow Flow LC-MS-Based Proteomic Profiling of Limited Samples Using Monolithic Capillary Columns and FAIMS Technology.

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.

SUBMITTER: Gregus M 

PROVIDER: S-EPMC7934643 | biostudies-literature | 2020 Nov

REPOSITORIES: biostudies-literature