A High-Throughput Workflow for High-Content Microplate-Based Assays via Mass Spectrometry Imaging

Abstract Microplate-based assays are indispensable in life sciences and drug discovery; however, conventional optical readouts provide limited chemical information. Here, we report a microplate-based mass spectrometry imaging (MP-MSI) workflow based on air flow-assisted desorption electrospray ionization (AFADESI) that enables high-throughput molecular analysis of complex biological samples with minimal sample consumption (down to 1 μL). In contrast to existing MSI-based high-throughput strategies that primarily perform single-mode screening, this platform integrates targeted quantitation and untargeted metabolomics from the same sample spot. Using formaldehyde (FA) as a model analyte, we developed a rapid spermidine derivatization method and performed full method validation in blank mouse plasma. The targeted assay achieved an interbatch precision of 6.18% RSD for the analyte-to-internal standard ratio across 0.01–0.8 mmol/L, with a minimum detectable concentration change of 1.12-fold. For untargeted metabolomics, the median within-run RSD evaluated from 180 repeated spottings of blank mouse plasma was 19.1%, corresponding to a minimum detectable fold change of 1.38, at a throughput of 2.2 min per sample. To demonstrate dual-mode integration, we applied the validated FA method to plasma from an Alzheimer’s disease mouse model (APP/PS1 and wild-type, 10 and 12 months of age), simultaneously quantifying FA and profiling the global metabolome from the same acquisition. Age- and genotype-dependent metabolic alterations were revealed. The platform was further extended to cell coculture models and to drug quantitation (e.g., irinotecan in plasma), demonstrating versatility across sample types and analytes. This integrated strategy offers a versatile platform for high-content screening in biomedical and pharmacological research.