Analyzing Assay Specificity in Metabolomics Using Unique Ion Signature Simulations

Targeted, untargeted, and data-independent acquisition (DIA) metabolomics workflows are often hampered by ambiguous identification based on either MS1 information alone or relatively few MS2 fragment ions. While DIA methods have been popularized in proteomics, it is less clear whether they are suitable for metabolomics workflows due to their large precursor isolation windows and complex coisolation patterns. Here, we quantitatively investigate the conditions necessary for unique metabolite detection in complex backgrounds using precursor and fragment ion mass-to-charge (m/z) separation, comparing three benchmarked mass spectrometry (MS) methods [MS1, MRM (multiple reaction monitoring), and DIA]. Our simulations show that DIA outperformed MS1-only and MRM-based methods with regards to specificity by factors of ∼2.8-fold and ∼1.8-fold, respectively. Additionally, we show that our results are not dependent on the number of transitions used or the complexity of the background matrix. Finally, we show that collision energy is an important factor in unambiguous detection and that a single collision energy setting per compound cannot achieve optimal pairwise differentiation of compounds. Our analysis demonstrates the power of using both high-resolution precursor and high-resolution fragment ion m/z for unambiguous compound detection. This work also establishes DIA as an emerging MS acquisition method with high selectivity for metabolomics, outperforming both data-dependent acquisition (DDA) and MRM with regards to unique compound identification potential.