Guidelines and considerations for building multidimensional libraries for untargeted MS-based metabolomics

Introduction: Feature annotation is crucial in untargeted metabolomics but remains a major challenge. The large pool of metabolites collected under various instrumental conditions is underrepresented in publicly available databases. Retention time (RT) and collision cross section (CCS) measurements from liquid chromatography ion mobility high-resolution mass spectrometers can be employed in addition to MS/MS spectra to improve the confidence of metabolite annotation. Recent advancements in machine learning focus on improving the accuracy of predictions for CCS and RT values. Therefore, high-quality experimental data are crucial to be used either as training datasets or as a reference for high-confidence matching.

Methods: This manuscript provides an easy-to-use workflow for the creation of an in-house metabolite library, offers an overview of alternative solutions, and discusses the challenges and advantages of using open-source software. A total of 100 metabolite standards from various classes were analyzed and subjected to the described workflow for library generation.

Results and discussion: The outcome was an open-access available NIST format metabolite library (.msp) with multidimensional information. The library was used to evaluate CCS prediction tools, MS/MS spectra heterogeneities (e.g., multiple adducts, in-source fragmentation, radical fragment ions using collision-induced dissociation), and the reporting of RT.