Biology Reference
In-Depth Information
its asymptomatic nature, we carried out
a search for diagnostic biomarkers that might
allow surgical intervention under non-life-
threatening conditions. Pooled human plasma
samples of 17 AAA and 17 control patients
were depleted of the most abundant proteins
and compared using PAcIFIC combined with
either spectral counting without TMT isobaric
tandem mass tags as described for the breast
cancer study
17
or as multiplexed qPAcIFIC
using TMTs. Both quantitative methods collec-
tively identi
range is highly increased with our PAcIFIC
strategy due to the fact that very narrow m/z
ranges are concentrated in the ion trap regard-
less of whether a precursor ion is present,
thus maximizing ion concentration prior to
CID. Importantly, using the latest and fastest
scanning ion traps available, an optimized
PAcIFIC method can be completed in less
than two days as opposed to the prior genera-
tion of ion traps. We have also demonstrated
how PAcIFIC may be multiplexed and quanti-
tative by use of TMT isobaric tandem mass
tags as well as by use of label-free spectral
counting. Additionally, a modi
ed a number of proteins as statisti-
cally differentially abundant between AAA and
control patients. Among differentially expressed
proteins, a subgroup of 19 was further selected
according to gene ontology classi
ed strategy
using high mass accuracy precursor ion scans
allowed the number of orphan peptides and
co-fragmenting peptides to be estimated. One
experiment that remains to be carried out is
a multiplexed PAcIFIC in which tandem mass
spectra are acquired at high resolution and
mass accuracy, which will allow more accurate
identi
cation and
implication in AAA for veri
cation by Western
blot (WB) in the same individual plasma
samples that comprised the pools. Five of
them
d
adiponectin, extracellular superoxide
dismutase, protein AMBP, kallistatin,
and
carboxypeptidase B2
d
were veri
ed to be
differentially upregulated in individual plasma
of AAA patients. One of the interesting observa-
tions was that spectral counting had a dynamic
range one order of magnitude (base 10) higher
than the TMT isobaric labeling data, implying
that spectral counting is better for quanti
cation of peptides in chimeric tandem
mass spectra. In an attempt to carry out
PAcIFIC analysis even faster, we recently
demonstrated proof of concept in a 24 hour
PAcIFIC using a captive spray ionization (CSI)
source (Bruker-Michrom, Auburn, CA)
that
cation
in a broader range of protein concentrations, but
obviously the TMT approach is required for
multiplexing. Plasma depletion of high abun-
dance proteins combined with quantitative
PAcIFIC analysis offered an ef
provides higher
flow rates and thus faster
regeneration of the analytical column between
injections. One of the principal advantages of
the PAcIFIC proteomic discovery strategy is
that it achieves a similar detectable dynamic
range as targeted MRM strategies. This ability
raises the possibility of a combined discovery-
validation experiment being carried out by
PAcIFIC, which we have recently tested using
a heavy isotope labeled peptide standard for
absolute quanti
cient and sensi-
tive tool for the screening of new potential
biomarkers of AAA.
18
PERSPECTIVES
cation of a target protein.
When the results from this nontraditional
PAcIFIC-based pseudo-MRM approach were
compared to results from a triple quadrupole
instrument, the data were in good agreement,
prompting further investigation of the concept
of combining discovery with validation in
a single platform.
We have shown that a systematic, data-
independent acquisition strategy can out-
perform standard data-dependent shotgun
proteomic methods, even with previously opti-
mized use of genome-based GPF for a similar
number of LC-MS/MS experiments. Dynamic
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