tsiklauri, g.; Zheng, R.; Kabella, N.; Prokofeva, P.; Pynn, C.; Kuster, B.

Current applications of mass spectrometry-based proteomics range from single cell to body fluid analysis that come with 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 very high-performance chromatographic separations in tandem with the high speed and sensitivity afforded by mass spectrometer. In this study, we focussed on the chromatographic angle and, more specifically, systematically evaluated proteome analysis performance across a wide range of chromatographic flow rates (0.3 - 50 L/min) and associated column diameters using a Vanquish Neo UHPLC coupled online to a Q Exactive HF-X mass spectrometer. Serial dilutions of HeLa cell line digests were used for benchmarking and total analysis time from injection-to-injection was intentionally fixed at 60 minutes (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 L/min) is a very robust, sensitive and quantitative alternative to nanoLC 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 application.