Biology Reference
In-Depth Information
MALDI-MS and MS/MS spectra of proteins and peptides.
The phenomenon of artifactual oxidation was recently demon-
strated by SL Cohen (
14
), whereby oxidation artifacts were
found on methionine and tryptophan residues.
10. A fully spotted stainless steel MALDI target plate should
contain several standard calibration spots which are needed for
mass calibration. Ideally, there are three MALDI spots (technical
replicates) made per sample in a sample spot set. Depending
on the fl uorescence intensity of the gel spot, either one or two
spots per sample are analyzed automatically using the MALDI
MS and automated MS/MS data acquisition options in which
the 10 top-intensity MS peaks are subjected to the subsequent
MS/MS analysis. If two spots are analyzed, the MS/MS
precursor ion masses from the fi rst spot are excluded from the
second spot analysis by using an “N fi rst precursors to skip”
option in the Interpretation Method of the instrument-control
software. Importantly, a MALDI-MS spectrum of a “digestion
blank” is acquired before the MS/MS data acquisition begins,
and the top background ions are placed on a permanent exclu-
sion list to stop the MALDI MS/MS software from acquiring
their MS/MS spectra. The MS/MS exclusion list should also
contain major human keratin and trypsin autolysis peaks. The
following set of instrument parameters can be applied to
enhance MS and MS/MS data quality for weaker 2D DIGE
spots processed using the dried-droplet MALDI spotting
method: total laser shots per spectrum/desorption spot is
~8,000/200 for the MS and ~8,000/100 for the MSMS; laser
intensity is fi xed at the level set to produce approximately
1-6 × 10
4
ion counts; and MS/MS acquisition stop conditions
are in effect after 10-15 consecutive subacquisitions from
desorption spots fail to pass acceptance criteria (S/N above
4-10) or after an accumulated spectrum reaches S/N of 50-80
with a minimum of 8-10 peaks above the threshold level and a
minimum of 15-20 subacquisitions accepted before the S/N
test stops. These MS/MS parameter ranges are set to preserve
sample while providing spectral quality by stopping the system
from acquiring poor quality MS/MS spectra which would
result in no peptide identifi cation, and to stop data acquisition
as soon as a good quality MS/MS spectrum has been collected.
In order to preserve samples further, a more laborious manual
stepwise procedure can be applied, in which the peptide mass
fi ngerprint (MS spectrum) is used to obtain an initial precursor
MS/MS ion list for targeted MS/MS verifi cation of the top
protein candidates. The best possible quality MS spectrum is
usually acquired fi rst, followed by the MS/MS spectra, if
possible, in the order of their importance to protein identifi ca-
tion. If faint gel spot peptide extracts fail to produce clear top
Search WWH ::
Custom Search