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

The thousands of features commonly observed when performing untargetedmetabolomics with quadrupole time-of-flight (QTOF) and Orbitrap massspectrometers often correspond to only a few hundred unique metabolitesof biological origin, which is in the range of what can be assayedin a single targeted metabolomics experiment by using a triple quadrupole(QqQ) mass spectrometer. A major benefit of performing targeted metabolomicswith QqQ mass spectrometry is the affordability of the instrumentsrelative to high-resolution QTOF and Orbitrap platforms. Optimizingtargeted methods to profile hundreds of metabolites on a QqQ massspectrometer, however, has historically been limited by the availabilityof authentic standards, particularly for unknownsthat have yet to be structurally identified. Here, we report a strategyto develop multiple reaction monitoring (MRM) methods for QqQ instrumentson the basis of high-resolution spectra, thereby enabling us to usedata from untargeted metabolomics to design targeted experiments withoutthe need for authentic standards. We demonstrate that using high-resolutionfragmentation data alone to design MRM methods results in the samequantitative performance as when methods are optimized by measuringauthentic standards on QqQ instruments, as is conventionally done.The approach was validated by showing that Orbitrap ID-X data canbe used to establish MRM methods on a Thermo TSQ Altis and two AgilentQqQs for hundreds of metabolites, including unknowns, without a dependenceon standards. Finally, we highlight an application where metaboliteprofiling was performed on an ID-X and a QqQ by using the strategyintroduced here, with both data sets yielding the same result. Thedescribed approach therefore allows us to use QqQ instruments, whichare often associated with targeted metabolomics, to profile knownsand unknowns at a comprehensive scale that is typical of untargetedmetabolomics.

Automated summary

Developing MRM methods on QqQ instruments based on high-resolution spectra allows for the design of targeted experiments using untargeted metabolomics data without the need for authentic standards, achieving the same quantitative performance as conventional methods using authentic standards. This approach enables profiling of hundreds of metabolites, including unknowns, without relying on standards, showcasing the potential for using QqQ instruments in a manner typical of untargeted metabolomics.