A Workflow to Perform Targeted Metabolomics at the Untargeted Scale on a Triple Quadrupole Mass Spectrometer

The thousands of
features commonly observed when performing untargeted
metabolomics with quadrupole time-of-flight (QTOF) and Orbitrap mass
spectrometers often correspond to only a few hundred unique metabolites
of biological origin, which is in the range of what can be assayed
in a single targeted metabolomics experiment by using a triple quadrupole
(QqQ) mass spectrometer. A major benefit of performing targeted metabolomics
with QqQ mass spectrometry is the affordability of the instruments
relative to high-resolution QTOF and Orbitrap platforms. Optimizing
targeted methods to profile hundreds of metabolites on a QqQ mass
spectrometer, however, has historically been limited by the availability
of authentic standards, particularly for “unknowns”
that have yet to be structurally identified. Here, we report a strategy
to develop multiple reaction monitoring (MRM) methods for QqQ instruments
on the basis of high-resolution spectra, thereby enabling us to use
data from untargeted metabolomics to design targeted experiments without
the need for authentic standards. We demonstrate that using high-resolution
fragmentation data alone to design MRM methods results in the same
quantitative performance as when methods are optimized by measuring
authentic standards on QqQ instruments, as is conventionally done.
The approach was validated by showing that Orbitrap ID-X data can
be used to establish MRM methods on a Thermo TSQ Altis and two Agilent
QqQs for hundreds of metabolites, including unknowns, without a dependence
on standards. Finally, we highlight an application where metabolite
profiling was performed on an ID-X and a QqQ by using the strategy
introduced here, with both data sets yielding the same result. The
described approach therefore allows us to use QqQ instruments, which
are often associated with targeted metabolomics, to profile knowns
and unknowns at a comprehensive scale that is typical of untargeted
metabolomics.