David A. Russo (@bluegreendr.bsky.social)

High-performance proteomics at any chromatographic flow rate 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 μL/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 μL/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. ### Competing Interest Statement R.Z. and C.P. are employees of Thermo Fisher Scientific. BK is a non-operational co-founder and shareholder of MSAID. The other authors declare no competing interests. * ABBREVIATIONS : ACN : Acetonitrile CAA : Chloroacetamide capLC : Capillary Liquid Chromatography CSF : Cerebrospinal Fluid CV : Coefficient of Variation DDA : Data-dependent Acquisition DIA : Data-Independent Acquisition DMSO : Dimethyl Sulfoxide DTT : Dithiothreitol EC50 : Effective Concentration to Reduce 50% of Protein Binding to Kinobeads FBS : Fetal Bovine Serum fmol : Femtomole FDR : False Discovery Rate FWHM : Full Width at Half Maximum HeLa : Human Cervical Carcinoma Cell Line HLB : Hydrophilic-Lipophilic Balance HPLC : High-Performance Liquid Chromatography i.d. : Internal Diameter Kdapp : Apparent Dissociation Constant LC : Liquid Chromatography LC-MS/MS : Liquid Chromatography-Tandem Mass Spectrometry µg : Microgram min : Minute µL : Microliter µLC : Microflow Liquid Chromatography mm : Milimeter mM : Milimolar MS : Mass Spectrometry ng : Nanogram nL : Nanoliter nLC : Nanoflow Liquid Chromatography nM : Nanomolar PSM : Peptide-spectrum Match RT : Retention Time SPD : Samples per Day SWATH-MS : Sequential Window Acquisition of All Theoretical Mass Spectra TFA : Trifluoroacetic Acid Å : Angstrom https://www.biorxiv.org/content/10.1101/2025.04.10.648108v1