Fundamental insights into Brønsted acid-induced adduction improve small metabolite profiling in mass spectrometry

Mass spectrometry (MS) is the primary tool to detect small metabolite ions for profiling bio-systems. However, small metabolite profiling in MS still needs to be improved and calls for new rules in ionization. Herein, we studied the Brønsted acid-induced multiple alkali metal cation adductions (BAI-mAMCA) in laser desorption/ionization MS, with annotation of BAI-mAMCA in complex bio-samples and improved profiling of small metabolites. Firstly, we engineered the enhanced mAMCA (relative intensities increased from 0.1–4.6 % to 76.1–89.9 %, p < 0.05) of small metabolites with desirable reproducibility via a nanoparticle-enhanced approach. Subsequently, we revealed the rule of BAI-mAMCA according to the proton dissociation ability of small metabolites qualitatively and quantitatively (|r| = 0.824–0.857), with validation using density functional theory calculation. Finally, we integrated a computational workflow of mAMCA-identity networking to annotate BAI-mAMCA in complex bio-samples and demonstrated excellent diagnostic performance (AUCs = 0.963–0.977) when applied to 300 serum samples from lung adenocarcinoma patients and healthy controls. We also achieved improved label-free quantitation (r² = 0.989–0.997, p < 0.05) and direct analysis of specific isomers based on the established BAI-mAMCA rules. This work provides both fundamental mechanisms and practical engineering strategies for advancing small metabolite profiling in MS, offering potential for integration into clinical diagnostic platforms.