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Additionally, having previously shown PAcIFIC
to be capable of detecting proteins an order
of magnitude (base 10) lower in abundance in
human serum than standard DDA methods,
we wished to better de
multiple times using all available adjacent
precursor ions. Although this approach has
a
flaw, in that any additional fragment ions not
belonging to the stated precursor ion in the
.dta
ne the detectable dy-
namic range of PAcIFIC. 12 To investigate these
ideas, we analyzed a Saccharomyces cerevisiae
whole-cell
file will decrease scores, it does not preclude
use to estimate chimeric spectra. Previously
we showed this to be an acceptable approach
using DDA data in which there are many
more precursor ions available than can be
selected. From these earlier DDA experiments
we estimated that approximately 8% of the
tandem mass spectra contained co-fragmenting
peptides. In the DIA data set acquired on a yeast
whole-cell lysate, we also found that approxi-
mately 8% of the peptide IDs could come from
these chimeric tandem mass spectra. It should
be expected that the rate of observing these acci-
dental CID or chimeric spectra would not
increase in going from DDA to DIA because
the only difference is that more tandem mass
spectra are acquired in a DIA experiment. Of
course these chimeric events are only valid for
the peptides where a precursor ion is observed
not for the orphan peptides in which, for the
moment, accounting for co-fragmenting orphan
peptides is not possible. Finally, we have previ-
ously shown that a high dynamic range (1E7)
could be achieved with this PAcIFIC technique
on a plasma sample that was an order of magni-
tude (base 10) better (at the same FDR) than
other published data using shotgun proteomic
methods, all of which used some sort of prefrac-
tionation method, such as strong cation
exchange (SCX) chromatography. 12 Weissmann
and colleagues reported on protein abundance
in S. cerevisiae, estimating that 80% of the pro-
teome is translated during normal growth condi-
tions with a dynamic range from fewer than 50
to more than 1
ed PAcIFIC
method that provided for acquisition of pre-
cursor ion scans at high mass accuracy while still
maintaining the basic PAcIFIC protocol. Specifi-
lysate using a modi
-
cally, a precursor ion mass spectrum was
acquired in the Orbitrap
mass analyzer simul-
taneous with the sequential acquisition of
ve
DIA tandem mass spectra in the LTQ. This
step provided three precursor ion spectra
equally distributed among a single set of each
15 DIA tandem mass spectra during one
PAcIFIC cycle. Because of the parallel nature of
the LTQ-Orbitrap mass spectrometer, these
precursor ion spectra could be acquired without
affecting the overall duty cycle of the PAcIFIC
method. The precursor ion mass spectra were
deconvoluted with Hardklör to extract a list of
all possible isotopic features. 20 From this type
of data acquisition and subsequent data anal-
ysis, two main bene
ts were derived. First, the
number of orphans could be precisely estimated.
Speci
cally, peptides were labeled as orphans
(1) when no precursor ion m/z value could be
detected in any of the adjacent precursor ion
mass spectra and (2) where no other precursor
ion corrected mass spectrum was detected that
matched the same database-derived peptide
sequence. By comparing all MS2 data with adja-
cent MS1 data, we could estimate that 18% of the
peptide IDs could be counted as orphan
peptides. Second, the number of accidental
CIDs or co-fragmenting peptides could also be
estimated by allowing each tandem mass spec-
tral .dta
10 6 protein molecules per
cell. 21 We have shown that unlike DDA
methods, the DIA PAcIFIC strategy is capable
of directly identifying proteins over the entire
dynamic range expressed by yeast under stan-
dard growth conditions. This feature makes
PAcIFIC capable of detecting yeast proteins
file (i.e., a single precursor ion mass
and list of associated fragment ions masses) to
be searched against the database using each of
the adjacent discovered precursor ions in turn.
Thus, each tandem mass spectrum was searched
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