The samples from each of the ten iTRAQ experiments were subjected to nano-LC
separation using an EASY-nLC Proxeon (Bruker Daltonics, Germany) coupled to a
Proteineer fc II (Bruker Daltonics, Germany) fraction collector as previously
described34 (link). For each iTRAQ experiment,
5 μl of respectively KCl fraction was
injected in triplicate. In total, 384 fractions were collected. The
UltrafleXtreme MALDI-TOF/TOF (Bruker Daltonics, Germany) instrument was operated
in the positive ion mode and controlled by the Compass for Flex software,
version 1.3 (FlexControl 3.3, FlexAnalysis 3.3, Bruker Daltonics, Germany). Five
thousand laser shots were accumulated per spectrum in the MS and MS/MS modes.
The spectrometric analysis was performed in automatic data-dependent mode. The
nonredundant precursor peptides were selected for MS/MS analysis using the
WARP-LC 1.3 software (Bruker Daltonics) with a signal-to-noise threshold of 12.
The MS spectra were externally calibrated using the Peptide Calibration Standard
mixture (Bruker Daltonics).
separation using an EASY-nLC Proxeon (Bruker Daltonics, Germany) coupled to a
Proteineer fc II (Bruker Daltonics, Germany) fraction collector as previously
described34 (link). For each iTRAQ experiment,
5 μl of respectively KCl fraction was
injected in triplicate. In total, 384 fractions were collected. The
UltrafleXtreme MALDI-TOF/TOF (Bruker Daltonics, Germany) instrument was operated
in the positive ion mode and controlled by the Compass for Flex software,
version 1.3 (FlexControl 3.3, FlexAnalysis 3.3, Bruker Daltonics, Germany). Five
thousand laser shots were accumulated per spectrum in the MS and MS/MS modes.
The spectrometric analysis was performed in automatic data-dependent mode. The
nonredundant precursor peptides were selected for MS/MS analysis using the
WARP-LC 1.3 software (Bruker Daltonics) with a signal-to-noise threshold of 12.
The MS spectra were externally calibrated using the Peptide Calibration Standard
mixture (Bruker Daltonics).
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