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

We introduce TRAM, a triple acquisition strategy on high-speed, time-of-flight mass spectrometry 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 ~ 1 second. 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. In this work, we deploy a workflow based on hydrophilic interaction chromatography (HILIC). For a broad panel of metabolites, we provide chromatographic retention times, and opti-mized conditions as a basis for targeted MRM experiments, listing accurate masses and sum formulas for fragment ions (including fully 13C labelled analogs). Validation experiments showed that TRAM offered (1) linear working ranges and limits of quantifica-tion 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. Acquiring up to 30 data-dependent spectra per MS cycle while still offering gold-standard absolute quantification, TRAM allows in-depth pro-filing and reduces required sample volume, time, cost, and environmental impact.