Capillary flow LC-MS (capLC-MS) with 100-300 μm inner diameter (ID) columns and flow rates from 1 to 10 μL/min provide increased MS sensitivity, lower solvent consumption, and reduced MS contamination compared with typical analytical flow LC-MS applications which runs at flow rates of more than 100 μL/min. Additionally, capLC-MS provides higher throughput in comparison to nano-flow LC, whilst affording a similar sensitivity achievable by loading higher sample amounts. Here we present a highly robust novel capillary-flow LC-MS platform that combines the Thermo Scientific™ capillary-flow UltiMate™ 3000 RSLCnano system (capLC), the new 150 μm ID ThermoScientific™ EASY-Spray™ column and the new Thermo Scientific™ Q Exactive™ HF-X mass spectrometer and describe how this novel capLC-MS platform can be used for high-throughput analysis, whilst providing higher MS sensitivity for routine shotgun proteomics due to the brighter ion-source of the Q Exactive HF-X mass-spectrometer.
Employing typical shotgun and targeted proteomics experiments (Full-scan MS, Data-Dependent Acquisition (DDA), and Parallel-Reaction Monitoring (PRM), the performance and robustness of
the novel capLC-MS platform was verified. We demonstrate that the novel capLC-MS platform is a sensitive and reliable solution for shotgun and targeted high-resolution accurate-mass (HRAM) proteomics experiments by combining the advantages of high-throughput analytical flow LC separations and the high sensitivity of nano-flow LC-MS analysis. Affording both robustness and sensitivity, this platform can be used for routine proteome profiling of complex samples including bio-fluids as well as targeted high-throughput quantification analysis of large sample cohorts.