High-Performance Proteomics Using Nano-, Capillary-, and Microflow Chromatographic Separations.

Current applications of mass-spectrometry-based proteomics range from single-cell to body fluid analysis, each presenting very different demands regarding sensitivity or sample throughput. Additionally, the vast molecular complexity of proteomes and the massive dynamic range of protein concentrations in these biological systems require highly performant chromatographic separations in tandem with the high speed and sensitivity afforded by modern mass spectrometers. In this study, we focused on the chromatographic aspect and, more specifically, systematically evaluated proteome analysis performance across a wide range of chromatographic flow rates (0.3-50 μL/min) and associated column diameters using a Vanquish Neo HPLC coupled online to a Q Exactive HF-X mass spectrometer. Serial dilutions of HeLa cell line digests were used for benchmarking, and the total analysis time from injection to injection was intentionally fixed at 60 min (24 samples per day). The three key messages of the study are that (i) all chromatographic flow rates are suitable for high-quality proteome analysis, (ii) capLC (1.5 μL/min) is a very robust, sensitive, and quantitative alternative to nLC for many applications, and (iii) showcased proteome, phosphoproteome, and drug proteome data provide sound empirical guidance for laboratories in selecting appropriate chromatographic flow rates and column diameters for their specific applications.