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.
Highlights
The field of proteomics aims at the qualitative and quantitative description of all proteins contained in complex biological samples
The most sensitive proteomics platforms are almost exclusively based on the combination of two key analytical methods: nanoflow highperformance liquid chromatography and tandem mass-spectrometry (MS/MS), hyphenated by electrospray ionization (ESI)
Despite the tremendous improvements in ultrasensitive nano-high-pressure liquid chromatography (HPLC)−ESI-MS/MSbased proteomics workflows and instrumentation, which capitalize on the massive increase in sample concentrations produced by extremely low elution volumes, the comprehensive characterization of, e.g., single mammalian cells still challenges the sensitivity of currently available technologies
Summary
The data revealed subtle, yet mechanistically insightful, differences on the separation characteristics of the two chromatographic systems compared in our study (please see Supplementary Figure 1) Most importantly, this analysis provides clear indications that the use of μPAC cartridges do allow for a more sensitive detection, of longer tryptic peptides, when compared to the pepMap columns (Supplementary Figure 1C). While 90% of all peptides identified with μPAC cartridges had a fwhm of
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