Practical considerations for optimizing untargeted metabolomics with Synapt XS HRMS: A tutorial

Metabolomics, the systematic study of small molecules in biological samples, has rapidly emerged as a key discipline for understanding metabolism across organisms. LCHRMS displays the dominant technique for global metabolomics due to its high resolution and fast detection. In addition, some instruments offer the option of separating analytes based on their mobility in a carrier gas (ion mobility separation). However, transferring existing protocols between instruments is often limited by differences in hardware, ion mobility capabilities, and instrument generations. In particular, traveling wave ion mobility spectrometry (TWIMS) can induce ion heating and fragmentation, primarily affecting labile polar metabolites (<500 Da), requiring individual adjustment of instrument settings. Because of component redesigns, parameters of previous systems cannot be directly transferred to the Synapt XS; this tutorial provides an overview of modifiable settings. We focus on the application of Synapt XS HRMS for untargeted metabolomics of polar and non-polar metabolites across diverse biological matrices. However, the procedures described for sample preparation, chromatographic separation, and data analysis are largely transferable to other high-performance devices. The tutorial includes step-by-step protocols for sample preparation, chromatographic separation of polar and non-polar compounds, adjustment of TWIMS parameters for labile metabolites, and the use of Progenesis QI, including creation of in-house spectral libraries and integration of existing fragment databases. Additionally, strategies for data acquisition modes and initial quality control and preprocessing are presented and discussed. Together, these methods provide a framework for highly sensitive, reproducible metabolomics analyses and, apart from MS-specific parameters, are broadly applicable to modern mass spectrometers from various manufacturers with comparable performance.