“Iridium Signature” Mass Spectrometric Probes: New Tools Integrated in a Liquid Chromatography–Mass Spectrometry Workflow for Routine Profiling of Nitric Oxide and Metabolic Fingerprints in Cells

Nitric oxide (NO) is a highly reactive signaling molecule involved in diverse biological processes. Simultaneous profiling of NO and associated metabolic fingerprints in a single assay allows more accurate assessments of cell states and offers the possibility to better understand its exact biological roles. Herein, a multiplexing LC–MS workflow was established for simultaneous detection of intracellular NO and various metabolites based on a novel “iridium signature” mass spectrometric probe (Ir-MSP841). This Ir-MSP841 can convert highly liable NO to a stable permanently charged triazole product (Ir-TP852), enabling direct MS detection of NO. This 191/193Ir-signature mass spectrometric probe-based approach is endowed with overwhelming advantages of interference-free, high quantitative accuracy, and great sensitivity (limit of detection down to 0.14 nM). It also reveals good linearity over a wide concentration range 12.5–500 nM and has been successfully employed for exploring the release behaviors of three representative NO donors in cells. Meanwhile, metabolic profiling results reveal that varying the concentrations of NO has distinct effects on various cellular metabolites. This study provides a robust, sensitive, and versatile method for simultaneous detection of NO and numerous metabolites in a single LC–MS run and expands its applications in biomedical research.