Untargeted Multiple Reaction Monitoring

Area of Science:

• Analytical Chemistry
• Biochemistry
• Mass Spectrometry

Background:

• Multiple reaction monitoring (MRM) offers sensitive and robust quantification but requires predefined transitions, limiting its application in discovery studies.
• Traditional MRM workflows necessitate extensive optimization and specific standards, posing challenges for large-scale or exploratory research.

Purpose of the Study:

• To introduce untargeted/micro/universal multiple reaction monitoring (uMRM), a novel workflow for automated generation of quantitative MRM methods.
• To enable the conversion of high-resolution untargeted liquid chromatography-mass spectrometry/MS (LC-MS/MS) data into scheduled triple-quadrupole MRM transitions.
• To provide a reproducible method for linking untargeted LC-MS/MS acquisition with targeted quantitation without authentic standards.

Main Methods:

• Utilized pooled-sample LC-MS and stepped-energy data-dependent acquisition (DDA) MS/MS for precursor and fragment ion information capture.
• Implemented automated deisotoping, in-source fragment filtering, and spline-based modeling of collision energy-dependent fragmentation.
• Scheduled transitions using retention times from pooled samples for deployment on triple-quadrupole instruments.

Main Results:

• Successfully generated quantitative MRM methods from untargeted discovery data across diverse biological matrices (urine, brain tissue, cultured cells).
• Demonstrated strong agreement between uMRM-derived and experimentally optimized collision energies across seven triple-quadrupole platforms.
• Validated the ability to deploy MRM transitions without nonlinear retention-time alignment or authentic standards.

Conclusions:

• uMRM provides a reproducible and automated approach to convert untargeted LC-MS/MS discovery data into quantitative MRM methods.
• This workflow significantly expands the utility of MRM for discovery-driven research by simplifying method development.
• uMRM facilitates the transition from broad-scale data acquisition to targeted, high-sensitivity quantitation.