Biology Reference
In-Depth Information
recent years for these purposes and have
greatly simpli
predictions for major instrument companies
that perform close to 92% on average to the
performance of the empirically derived collision
energy in MRM-MS assays, as reported in
a recent study.
52
Final results generated by
Skyline can be exported into tabular format
that is compatible with Microsoft Excel
ed the efforts needed to set up
and optimize MRM-MS assays as well as data
processing after acquisition.
All major mass spectrometer companies
have software either developed speci
cally
for MRM-MS assays or adapted to handle
MRM-MS data. The commercial software appli-
cations from the major mass spectrometer com-
panies are Pinpoint
for
easy statistical analysis. As Skyline seems to
have the capabilities for method design for tar-
geted MRM-MS in combination with isotope-
labeled internal standards, support for assay
optimization, and automated data analysis on
all the major instrument platforms, it would be
a great tool for biomarker veri
(Thermo Fisher Scienti
c),
MultiQuant
(AB Sciex), MassHunter
(Agilent
Technology), and TargetLynx
(Waters). Pin-
point and MultiQuant are the most commonly
used commercial software, which is not
surprising, as the TSQ instrument from Thermo
and the QTRAP instrument from AB Sciex are
most commonly used for targeted peptide
MRM-MS experiments. Both MultiQuant and
Pinpoint have capability for MRM-MS method
design, assay optimization, and data processing
for experiments and data generated by their
speci
cation studies
when multisite studies are being conducted.
In addition to software tools developed to
facilitate MRM-MS assay design and data
processing automation, efforts have also been
made to address inaccurate and imprecise quan-
ti
cation due to interferences or inconsistencies
in MRM-MS signals, which is especially impor-
tant when dealing with such complex sample
matrices as plasma and serum. An algorithm-
denoted automated detection of inaccurate
and imprecise transitions (AuDIT) has been
developed to automatically identify potential
problematic data to be manually inspected and
corrected. The program demonstrated better
than 94% identi
c instruments.
The commonly used open source software
includes Skyline and MRMer.
50,51
MRMer takes
data in the platform-independent mzXML data
format and allows data extraction, visualization,
and analysis. Among the software choices,
Skyline uses data from all major instrument
companies in their native form and to design
targeted MRM-MS assays directly exported in
speci
cation accuracy of errant data
with high sensitivity and speci
city.
53
mProphet
is another automated data processing software
developed with a probabilistic scoring model
where decoy transitions must be measured
with target transitions for statistical validation
of MRM-MS data,
54
which could be essential in
large-scale biomarker veri
c major instrument method forms. When
deciding on peptides and transitions to monitor
for biomarker veri
cation, previous search
results from biomarker discovery stage, public
available spectral library, and in silico predic-
tions could all be used in the decision-making
process. Skyline also fully supports quantitative
data analysis using isotope-labeled internal
standards. Collision energy is one of the instru-
ment parameters that is often optimized for
each peptide to achieve best signal intensity,
which could be expensive in terms of both time
and resources, especially when a large amount
of peptides are involved. Skyline also provides
instrument platform-dependent collision energy
cation studies where
manually checking data is impractical.
A software application called ATAQS was
also developed in which all the steps from
MRM-MS assay design, assay re
nement, assay
optimization, decoy transition generation, data
processing, and statistical data validation are
all integrated into one package.
55
Its perfor-
mance, although implemented for large-scale
MRM-MS-based peptide quantitation in complex
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