Multiplexed Targeted Spatial Mass Spectrometry Imaging Assays to monitor lipids and NAD+ metabolites in CD38 knockout mice exhibiting improved metabolism.

Mass spectrometry imaging (MSI) is a rapidly advancing technology that provides mapping of the spatial molecular landscape of tissues for a variety of analytes. Matrix-assisted laser desorption/ionization (MALDI)-MSI is commonly employed, however, confident in situ identification and accurate quantification of analytes remain challenging. We present a novel imaging methodology combining trapped ion mobility spectrometry (TIMS)-based parallel accumulation-serial fragmentation (PASEF) with MALDI ionization for targeted imaging parallel reaction monitoring (iprm-PASEF). We investigated the spatial distribution of lipids and metabolites in liver tissues from wild-type and CD38 knockout mice (CD38^-/-). CD38, an enzyme involved in nicotinamide adenine dinucleotide (NAD+) metabolism, significantly influences liver metabolic function and contributes to age-related NAD+ decline. Although CD38 deletion previously was linked to improved metabolic phenotypes, the underlying spatial metabolic mechanisms are poorly understood. The spatial iprm-PASEF workflow enabled confident identification and differentiation of lipid isomers at the MS2 fragment ion level and revealed increased NAD⁺ and decreased adenosine diphosphate ribose (ADPR), a by-product of NAD⁺ hydrolysis, in CD38^-/- livers. This approach provided confident, specific, and robust MS2-based identification and quantification of fragment ions in spatial MSI experiments. Additionally, the innovative iprm-PASEF opens unprecedented opportunities for spatial metabolomics and lipidomics, offering spatially resolved insights into molecular mechanisms.