A well-ordered nanoflow LC-MS/MS approach for proteome profiling using 200 cm long micro pillar array columns

In bottom-up proteomics, capillaries up to 75 cm long with internal diameters of 50 to 100 {micro}m packed with sub-2-{micro}m C18-functionalized particles are routinely used in combination with high-resolution mass spectrometry. Unlike such conventional liquid chromatography (LC) columns, micro pillar array columns ({micro}PAC) are fabricated using micromachining technology, resulting in perfectly ordered chromatographic separation beds, leading to a minimized analyte dispersion while column permeability is increased by one order of magnitude. This allows using very long columns (up to 200 cm) at only a fraction of the pressure needed to operate packed bed columns. To validate {micro}PAC column performances, different amounts of tryptic digests of HEK293T cell lysates were prepared and separated using a 200 cm {micro}PAC column or a 40 cm long conventional column. Using an Orbitrap Elite instrument, on average 25% more proteins were identified with the {micro}PAC column. Moreover, the rate at which the peak width increases with gradient time is much lower on the {micro}PAC column. For a 10-hour long gradient, average peak widths below 0.5 min were observed, resulting in consistent identification of over 5,000 proteins. Combining long solvent gradients and this new type of LC column, substantial improvements in proteome coverage could be obtained. Finally, we demonstrated high reproducibility and durability of the {micro}PAC column. Data are available via ProteomeXchange with identifiers PXD011547 and PXD013235.