In the past few years, micro pillar array column technology (µPAC™) has evolved from a tool to perform fundamental LC research to a powerful alternative for classical nano LC columns. The inherent high permeability and low ‘on-column’ dispersion obtained by the perfect order of the separation bed makes pillar array based chromatography unique in its kind. The peak dispersion originating from heterogeneous flow paths in the separation bed is drastically reduced and therefore components remain much more concentrated during separation (1). Apart from an improved efficiency, pillar array columns can also be designed with substantially lower flow resistances compared to packed bed columns.
Using a 50 cm long reversed phase C18 µPAC™ nano LC column in combination with a Thermo Orbitrap Elite mass spectrometer for detection, we demonstrate extremely robust and high-throughput proteome analysis at capillary flow rates up to 2 µl/min. At these flow rates, sample throughput can be increased to 20, 30, 60 and even 100 samples per day with corresponding peak capacity values (2) of respectively 300, 250, 200 and 150. When comparing the chromatographic performance that could be obtained for single protein and cell lysate tryptic digest samples to state-of-the art packed bed nano LC columns (packed with sub 2 µm silica particles), average peptide peak widths could be reduced by a factor of 1.6. For complex HeLa cell digest samples, this resulted in an increase in peptide and protein group identifications of respectively 60 and 40%.
To increase the throughput even further, a micro pillar array based trapping column was implemented into the workflow. By doing so, sample loading times for diluted protein digest samples could be reduced up to a factor of 15 without affecting the chromatographic performance.