Conventional-Flow Liquid Chromatography–Mass Spectrometry for Exploratory Bottom-Up Proteomic Analyses

Due to its sensitivity and productivity, bottom-up proteomics based on liquid chromatography–mass spectrometry (LC–MS) has become the core approach in the field. The <i>de facto</i> standard LC–MS platform for proteomics operates at sub-μL/min flow rates, and nanospray is required for efficiently introducing peptides into a mass spectrometer. Although this is almost a “dogma”, this view is being reconsidered in light of developments in highly efficient chromatographic columns, and especially with the introduction of exceptionally sensitive MS instruments. Although conventional-flow LC–MS platforms have recently penetrated targeted proteomics successfully, their possibilities in discovery-oriented proteomics have not yet been thoroughly explored. Our objective was to determine what are the extra costs and what optimization and adjustments to a conventional-flow LC–MS system must be undertaken to identify a comparable number of proteins as can be identified on a nanoLC–MS system. We demonstrate that the amount of a complex tryptic digest needed for comparable proteome coverage can be roughly 5-fold greater, providing the column dimensions are properly chosen, extra-column peak dispersion is minimized, column temperature and flow rate are set to levels appropriate for peptide separation, and the composition of mobile phases is fine-tuned. Indeed, we identified 2 835 proteins from 2 μg of HeLa cells tryptic digest separated during a 60 min gradient at 68 μL/min on a 1.0 mm × 250 mm column held at 55 °C and using an aqua–acetonitrile mobile phases containing 0.1% formic acid, 0.4% acetic acid, and 3% dimethyl sulfoxide. Our results document that conventional-flow LC–MS is an attractive alternative for bottom-up exploratory proteomics