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Anal Chem. 2019 Nov 19;91(22):14203-14207. doi: 10.1021/acs.analchem.9b02899. Epub 2019 Oct 31.

Improved Sensitivity in Low-Input Proteomics Using Micropillar Array-Based Chromatography.

Author information

1
IMBA-Institute of Molecular Biotechnology of the Austrian Academy of Sciences , Dr. Bohr Gasse 3 , A-1030 Vienna , Austria.
2
IMP-Institute of Molecular Pathology , Campus-Vienna-Biocenter 1 , A-1030 Vienna , Austria.
3
PharmaFluidics , Technologiepark-Zwijnaarde 82 , B-9052 Gent , Belgium.
4
Vrije Universiteit Brussel , Department of Chemical Engineering , Pleinlaan 2 , B-1050 Brussels , Belgium.
5
Department of Medical Genetics, Life Sciences Institute , University of British Columbia , Vancouver Campus, 2350 Health Sciences Mall , Vancouver , British Columbia , Canada , V6T 1Z3.

Abstract

Capitalizing on the massive increase in sample concentrations which are produced by extremely low elution volumes, nanoliquid chromatography-electrospray ionization-tandem mass spectrometry (nano-LC-ESI-MS/MS) is currently one of the most sensitive analytical technologies for the comprehensive characterization of complex protein samples. However, despite tremendous technological improvements made in the production and the packing of monodisperse spherical particles for nanoflow high-pressure liquid chromatography (HPLC), current state-of-the-art systems still suffer from limits in operation at the maximum potential of the technology. With the recent introduction of the μPAC system, which provides perfectly ordered micropillar array based chromatographic support materials, completely new chromatographic concepts for optimization toward the needs of ultrasensitive proteomics become available. Here we report on a series of benchmarking experiments comparing the performance of a commercially available 50 cm micropillar array column to a widely used nanoflow HPLC column for the proteomics analysis of 10 ng of tryptic HeLa cell digest. Comparative analysis of LC-MS/MS-data corroborated that micropillar array cartridges provide outstanding chromatographic performance, excellent retention time stability, and increased sensitivity in the analysis of low-input proteomics samples and thus repeatedly yielded almost twice as many unique peptide and unique protein group identifications when compared to conventional nanoflow HPLC columns.

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