ac5b00708_si_001.pdf (1.7 MB)
Comprehensive Two-Dimensional Liquid Chromatography with Stationary-Phase-Assisted Modulation Coupled to High-Resolution Mass Spectrometry Applied to Proteome Analysis of Saccharomyces cerevisiae
journal contribution
posted on 2015-05-19, 00:00 authored by Rudy J. Vonk, Andrea F. G. Gargano, Ekaterina Davydova, Henk L. Dekker, Sebastiaan Eeltink, Leo J. de Koning, Peter
J. SchoenmakersStationary-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.