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precursor ion m/z range of 400 to 1,400 u is
covered (
Figure 1
B). The acquired tandem mass
spectra are then matched to peptide sequence in
a database using the center of the precursor isola-
tion window as the parent ion mass in each
tandem mass spectrum
peptides with low or no detectable precursor ion
signal are selected for CID.
12
Recent Improvements to PAcIFIC
To date, the PAcIFIC strategy has been used
primarily on ion trap mass spectrometers from
a single manufacturer (Thermo Scienti
file and considering
charge states of 2
. Therefore, a relatively
large precursor mass tolerance of
and 3
þ
þ
/
e
3.75
c, Wal-
tham, MA), but it will work equally well on other
ion-trapping-style instruments. Since the initial
PAcIFIC report, ion trapping ef
þ
Daltons, corresponding to
/
e
1.25 m/z unit
for a triply charged precursor ion, is used to
account for any precursor that could physically
be present in the trap after isolation (
Figure 1
C).
Much larger precursor ion isolation windows
have historically been used for data-independent
analysis, providing the advantage of fewer injec-
tions to cover the same precursor ion m/z
range.
13
e
15
However, the resulting complexity of
these tandem mass spectra made use of special
algorithms to reconstruct the precursor ion/frag-
ment ion lineage necessary. The narrow isolation
window used for CID in PAcIFIC determines,
by default, the peptide
þ
ciency and scan
speed from Thermo have steadily improved,
which has allowed the number of m/z channels
that may be conducted in a single LC-MS/MS to
increase. This upgrade has resulted in subsequent
decreases in the total number of hours needed to
complete a full PAcIFICexperiment from the orig-
inal
five days to only three days on an LTQ XL
(Thermo Scienti
c) and now down to less than
two days on the LTQ Velos (Thermo Scienti
c)
without affecting peptide identi
cation rates.
The original PAcIFIC strategy used a DIA tandem
mass spectrometry (MS2) cycle consisting of 10
events incremented by 1.5 u from one m/z
channel to the next scan event (i.e., 400.0, 401.5,
403.0, etc.). This process required approximately
67 LC-MS/MS injections, each covering 15 m/z,
or
s precursor ion mass
without need for reconstruction of the lineage or
accuratemassof theprecursor allowingastandard
database search routine to make sequence assign-
ments. Although subsequent versions of PAcIFIC
may use accurate mass and often acquire MS1
scans, this initial version was designed to work
solely on a low-resolution ion trap with standard
database search engines. Thus, the initial report
showed that compared to our DDA GPF based
strategy, theDIAPAcIFIC strategy easily doubled
protein identi
'
100 min LC
gradient) on an LTQ XL ion trap to accomplish
a full range analysis from 400 to 1,400 u. In order
to explore howbest tominimize the time required
without a decrease in peptide identi
five days (i.e., 67 injections
cations,
several experiments were performed in which
three essential parameters were modi
cations as well as increased indi-
vidual protein sequence coverage by 10%.
12
Addi-
tionally, as shown by our initial work on serum,
the dynamic range was also increased by at least
one order of magnitude (base 10) because (1) the
DIA nature of this strategy is not biased by the
presence or absence of precursor ion signals, (2)
acquisition of a tandem mass spectrum from
a narrowly selected m/z range, which is opposite
to the broad range used for most DDA experi-
ments,
ed: (1)
number of MS2 scan events per cycle, (2)
precursor ion isolation width, and (3) m/z
channel increment. For this optimization, the
same Pseudomonas aeruginosa digest used in the
original publication was used.
12
A DIA MS2
scan being on average 0.3 seconds on an LTQ XL
ion trap, only a limited number of scan events
can be programmed, which should not exceed
the average chromatographic peak retention
time (approx. 10
e
15 seconds). Ideally, at least
two to three of these cycles should be performed
fills the trap only with ions of interest for
CID ensuring higher data quality than would be
possible from a DDA experiment, and (3) even
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