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 (2D-LC or LC × LC) can coupled to MS. Low-flow separations are attractive for HRMS analysis, but they tend lengthy. In this work, low-flow, online, actively modulated system, based on hydrophilic-interaction (HILIC) first dimension reversed-phase (RPLC) second dimension, was developed separate mixtures peptides. Miniaturization permitted analysis (1–5 ?g) coupling with micro-ESI MS (1 ?L min?1). All components were focused automatically transferred from HILIC RPLC using stationary-phase-assisted active modulation (C18 traps) deal solvent-incompatibility dilution issues. Optimization setup performed columns provide more efficient separation higher identification rates than obtained one-dimensional (1D) LC. A 60% increase peak capacity 2D compared 1D-RPLC 17–34% number proteins identified achieved (2D-yeast-8280 peptides 2D-kidney tissue-8843 peptides), without increasing time (2 h).