Development of comprehensive two-dimensional low-flow liquid- chromatography setup coupled to high-resolution mass spectrometry for shotgun proteomics

Bottom-up proteomics provides often small amounts of highly complex samples that cannot be analysed by direct mass spectrometry (MS). To gain a better insight in the sample composition, liquid chromatography (LC) and (comprehensive) two-dimensional liquid chromatography (2D-LC or LC x LC) can be coupled to the MS. Low-flow separations are attractive for HRMS analysis, but they tend to be lengthy. In this work, a low-flow, online, actively modulated LC x LC system, based on hydrophilic-interaction liquid chromatography (HILIC) in the first dimension and reversed-phase liquid chromatography (RPLC) in the second dimension, was developed to separate complex mixtures of peptides. Miniaturization permitted the analysis of small sample amounts (1-5 µg) and direct coupling with micro-ESI MS (1 µL min^-1). All components were focused and automatically transferred from HILIC to RPLC using stationary-phaseassisted active modulation (C18 traps) to deal with solvent-incompatibility or dilution issues. Optimization of the setup was performed for the HILIC columns and the RPLC columns to provide a more efficient separation and higher identification rates than obtained using one-dimensional (1D) LC. A 60% increase in peak capacity was obtained with the 2D setup compared to a 1D-RPLC separation and a 17