DOI: 10.1101/472134Nov 19, 2018Paper

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

BioRxiv : the Preprint Server for Biology
Jeff Op De BeeckFrancis Impens

Abstract

In bottom-up proteomics, capillaries up to 75 cm long with internal diameters of 50 to 100 μm packed with sub-2μm C18-functionalized particles are routinely used in combination with high-resolution mass spectrometry. Unlike such conventional liquid chromatography (LC) columns, micro pillar array columns (μPAC™) 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 μPAC™ column performances, different amounts of tryptic digests of HEK293T cell lysates were prepared and separated using a 200 cm μPAC™ column or a 40 cm long conventional column. Using an Orbitrap Elite instrument, on average 25% more proteins were identified with the μPAC™ column. Moreover, the rate at which the peak width increases with gradient time is much lower on the μPAC™ 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...Continue Reading

Related Concepts

Blood Capillaries
Liquid Chromatography
Digestion
Mass Spectrometry
Pillar Cell
Dispersion
Instrument - Device
Addition to Lower Extremity, Patten Bottom
Particle
Chromatography Systems, Liquid, Packed Column

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