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across a chromatographic peak in order to obtain
a tandem mass spectrum on a given ion at the
highest possible base peak intensity to yield at
least one high-quality tandem mass spectrum for
that ion. After a series of such optimization exper-
iments, when the results were compared to the
original publication results, it was found that
comparable protein and peptide hits could be
achieved with a combination of 15 MS2 spectra,
an isolationwidth of 2.5m/z, and a channel incre-
ment of 1.5 m/z instead of the original 10 MS2
spectra.
16
This combination represents a 33%
improvement in analysis time by going from 4.7
days to 3.1 days (45 injections
PAcIFIC experiment over 1,000 u to approxi-
mately 1.9 days (27 injections
100 min LC
gradient).
PAc
IFIC AND QUANTIFICATI
ON
Quantitative PAcIFIC (qPAcIFIC)
We have recently shown that spectral counting
can be used with standard PAcIFIC data.
17,18
The
ease of label-free spectral counting makes
PAcIFIC immediately available for quantitative
proteomics. We also investigated the coupling
of the PAcIFIC strategy with the stable isotope
isobaric tagging approaches, such as with the
tandem mass tag (TMT) method.
19
In order to
circumvent the one-third m/z cutoff limit
inherent to ion trap instruments, the TMT quanti-
tative reporter ions could not be recorded in
a regular tandemmass spectrum, so a supplemen-
tary pulsed q dissociation (PQD) mass spectral
scan was added to the PAcIFIC DIA strategy to
record these ions in the low m/z region.
Figure 2
illustrates the acquisition process (A) as well as
data processing (B) used for the TMT-based quan-
titative PAcIFIC (qPAcIFIC) strategy. In order to
keep the duty cycle (time to go through a speci
100 min LC
gradient). Additionally, using 20MS2 scanevents,
only a slight decrease in the number of peptide
identi
cations was observed, but such losses
may be acceptable should time be limited. In this
latter case, only 2.4 days (34 injections
100 min
LC gradient) are necessary to cover an identical
m/z range. Interestingly, increasing the channel
increment from 1.5 to a higher value resulted in
a drastic loss inpeptide identi
cations.We believe
this loss occurs because an overlap of the
precursor ion isolation width for CID between
each m/z channel of at least 33% is optimum for
matching peptide isotopic envelopes and there-
fore maximizing the number of high-quality
tandem mass spectra. This overlap in m/z chan-
nels used for PAcIFIC prevents isotopic distribu-
tions from being split between the channels,
which results in a decrease in the quality of result-
ing tandem mass spectra. We can also speculate
that this effect may be exacerbated by the use of
ion trap isolation waveforms that truncate on
either side of the selected value as expected.
Finally, using one of the latest dual linear ion traps
(i.e., an LTQ Velos
ed
number of MS2 scan event cycle) to a reasonable
time (i.e., 5.4 seconds on average) using an opti-
mized 15-scan event cycle as previously dis-
cussed, PQD spectra were acquired on only
a limited range corresponding to the region
where the isobaric tags are expected (121
e
132
m/z).
16
The information contained in these pairs
of CID-PQD mass spectra was then combined
into a composite tandem mass spectrum that
was used for database searching as well as
quanti
), where the overall tandem
mass spectral acquisition rate is approximately
0.1 second faster than that of the prior generation
LTQ XL, allows a single PAcIFIC cycle to be
stretched to 25 MS2 spectra without degradation
in protein identi
cation. When the qPAcIFIC experi-
ments were applied to a mixture of 14 protein
standards varying concentrations of each pro-
tein, experimental ratios were in accordance
with the expected ranking of the theoretical
data. Additionally, when the qPAcIFIC approach
was applied to a more complex cell lysate of
cations over the original 15
MS2 spectra. This increased scan rate perfor-
mance decreases the time needed for a full
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