Triple acquisition mass spectrometry (TRAM) combining targeted and non-targeted metabolomics in a single run

Background: We introduce TRAM, a triple acquisition strategy on a high-speed quadrupole time-of-flight mass spectrometer for merging non-targeted and targeted metabolomics into one run. TRAM stands for "quasi-simultaneous" acquisition of (1) a full scan MS1, (2) top 30 data-dependent MS2 (DDA), and (3) targeted scheduled MS2 for multiple reaction monitoring (MRM) within measurement cycles of similar to 1 s. TRAM combines the selectivity and sensitivity of state-of-the-art targeted MRM-based methods with the full scope of non-targeted analysis enabled by high-resolution mass spectrometry. Results: In this work, we deploy a workflow based on hydrophilic interaction liquid chromatography (HILIC). For a broad panel of metabolites, we provide chromatographic retention times, and optimized conditions as a basis for targeted MRM experiments, listing accurate masses and sum formulas for fragment ions (including fully C-13 labeled analogs). Validation experiments showed that TRAM offered (1) linear working ranges and limits of quantification comparable to MRM-only methods, (2) enabled accurate quantification in SRM 1950 human plasma reference material, and (3) was equivalent to DDA-only approaches in non-targeted metabolomics. Metabolomics in human cerebrospinal fluid showcased the power of the strategy, emphasizing the need for high coverage/high throughput metabolomics in clinical studies. Significance: Acquiring up to 30 data-dependent spectra per MS cycle while still offering gold standard absolute quantification down to low nanomolar concentrations, TRAM allows in-depth profiling and reduces required sample volume, time, cost, and environmental impact.