期刊
ANALYTICA CHIMICA ACTA
卷 1156, 期 -, 页码 -出版社
ELSEVIER
DOI: 10.1016/j.aca.2021.338349
关键词
Nano two-dimensional liquid chromatography; Hydrophilic interaction liquid chromatography; Active modulation; Peptide separation
资金
- Horizon 2020 -Excellent Science -European Research Council (ERC) [694151]
- Netherlands Organization for Scientific Research, NWO Veni grant IPA [722.015.009]
Bottom-up proteomics often deals with small amounts of complex samples that cannot be directly analyzed by mass spectrometry. To better understand sample composition, liquid chromatography (LC) and two-dimensional liquid chromatography (2D-LC) can be combined with mass spectrometry. In this study, a low-flow, actively modulated LC x LC system was developed to separate complex peptide mixtures, allowing for efficient analysis of small sample amounts and improved peak capacity compared to one-dimensional separation.
Bottom-up proteomics provides often small amounts of highly complex samples that cannot be analysed by direct mass spectrometry (MS). To gain a better insight in the sample composition, liquid chromatography (LC) and (comprehensive) two-dimensional liquid chromatography (2D-LC or LC x LC) can be coupled to the MS. Low-flow separations are attractive for HRMS analysis, but they tend to be lengthy. In this work, a low-flow, online, actively modulated LC x LC system, based on hydrophilic-interaction liquid chromatography (HILIC) in the first dimension and reversed-phase liquid chromatography (RPLC) in the second dimension, was developed to separate complex mixtures of peptides. Miniaturization permitted the analysis of small sample amounts (1-5 mu g) and direct coupling with micro-ESI MS (1 mu L min(-1)). All components were focused and automatically transferred from HILIC to RPLC using stationary-phaseassisted active modulation (C18 traps) to deal with solvent-incompatibility or dilution issues. Optimization of the setup was performed for the HILIC columns and the RPLC columns to provide a more efficient separation and higher identification rates than obtained using one-dimensional (1D) LC. A 60% increase in peak capacity was obtained with the 2D setup compared to a 1D-RPLC separation and a 17
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