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Comprehensive Two-Dimensional Liquid Chromatography with Stationary-Phase-Assisted Modulation Coupled to High-Resolution Mass Spectrometry Applied to Proteome Analysis of Saccharomyces cerevisiae

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University of Amsterdam, van’t Hoff Institute for Molecular Sci, Analytical-Chemistry Group, and §Swammerdam Institute for Life Sciences, Mass Spectrometry of Biomacromolecules, Science Park 904, 1098 XH Amsterdam, The Netherlands
TI-COAST, Science Park 904, 1098 XH Amsterdam, The Netherlands
Vrije Universiteit Brussel, Department of Chemical Engineering, Pleinlaan 2, B-1050 Brussels, Belgium
*Tel.: +31 (0)20 525 6424. Fax: +31 (0)20 525 5604. E-mail: [email protected]
Cite this: Anal. Chem. 2015, 87, 10, 5387–5394
Publication Date (Web):April 20, 2015
Copyright © 2015 American Chemical Society

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    Stationary-phase-assisted modulation is used to overcome one of the limitations of contemporary comprehensive two-dimensional liquid chromatography, which arises from the combination of a first-dimension column that is typically narrow and long and a second-dimension column that is wide and short. Shallow gradients at low flow rates are applied in the first dimension, whereas fast analyses (at high flow rates) are required in the second dimension. Limitations of this approach include a low sample capacity of the first-dimension column and a high dilution of the sample in the complete system. Moreover, the relatively high flow rates used for the second dimension make direct (splitless) hyphenation to mass spectrometry difficult. In the present study we demonstrate that stationary-phase-assisted modulation can be implemented in an online comprehensive two-dimensional LC (LC × LC) setup to shift this paradigm. The proposed active modulation makes it possible to choose virtually any combination of first- and second-dimension column diameters without loss in system performance. In the current setup, a 0.30 mm internal diameter first-dimension column with a relatively high loadability is coupled to a 0.075 mm internal diameter second-dimension column. This actively modulated system is coupled to a nanoelectrospray high-resolution mass spectrometer and applied for the separation of the tryptic peptides of a six-protein mixture and for the proteome-wide analyses of yeast from Saccharomyces cerevisiae. In the latter application, about 20000 MS/MS spectra are generated within 24 h analysis time, resulting in the identification of 701 proteins.

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