Multidimensional performance assessment of micro pillar array column chromatography combined to ion mobility-mass spectrometry for proteome research.

Micro pillar arrays columns (μPAC) are recent nanoflow liquid chromatographic (LC) systems featuring highly ordered pillars containing an outer porous shell grafted with C18 groups. This format limits backpressure and allows the use of extremely long separation channel (up to 2 m). In this study, we evaluated the use of μPAC in combination with ion mobility mass spectrometry (IM-MS). In IM-MS, ions are separated in gas-phase based on their size and charge. μPAC was compared to two other nanoflow systems and a state-of-the-art ultra-high-pressure liquid chromatograph (UHPLC). Performances in the four dimensions of information (LC, IM, MS and intensity) were calculated to assess the multidimensional efficiency of each tested system. μPAC proved to be superior to other nanoflow systems by producing more efficient peaks regardless of the gradient time employed which resulted in higher peak capacities (386 after 240 min gradient). In combination with IM, 3 times more peaks could be separated without loss of analysis time. Although UHPLC-ESI was superior from a chromatographic point of view, its sensitivity was rather limited compared to nanoflow LCs. On average, peaks in μPAC were 45-times more intense. Finally, μPAC combined to IM prove to enhance the proteome coverage by identifying two times more peptides than nanoflow LCs and ten times more than UHPLC. As a conclusion, μPAC combined to IM seems to be a suitable platform for discovery proteomics due to its high separation capacities.