PANAMA-enabled high-sensitivity dual nanoflow LC-MS metabolomics and proteomics analysis

High-sensitivity nanoflow liquid chromatography (nLC) is seldom employed in untargeted metabolomics because current sample preparation techniques are inefficient at preventing nanocapillary column performance degradation. Here, we describe an nLC-based tandem mass spectrometry workflow that enables seamless joint analysis and integration of metabolomics (including lipidomics) and proteomics from the same samples without instrument duplication. This workflow is based on a robust solid-phase micro-extraction step for routine sample cleanup and bioactive molecule enrichment. Our method, termed proteomic and nanoflow metabolomic analysis (PANAMA), improves compound resolution and detection sensitivity without compromising the depth of coverage as compared with existing widely used analytical procedures. Notably, PANAMA can be applied to a broad array of specimens, including biofluids, cell lines, and tissue samples. It generates high-quality, information-rich metabolite-protein datasets while bypassing the need for specialized instrumentation. [Display omitted] •Optimized workflow enables metabolomics and proteomics using single instrumentation•PANAMA enables nanoflow LC by adapting solid-phase micro-extraction for metabolomics•PANAMA enhances chromatography separation, sensitivity, and resolution for metabolomics•PANAMA identifies metabolites and proteins in same sample without sacrificing coverage The ability to routinely, sensitively, and reproducibly analyze both cellular proteins and metabolite mixtures from the same biospecimens can enhance the discovery of biomolecules associated with basic biochemical processes and pathobiological states. Yet existing mass spectrometry-based profiling methods rely on specialized protocols and duplicated instrumentation platforms, resulting in increased time, sample consumption, and costs. We sought to generate an effective platform for both metabolomics and proteomics studies on the same samples by enabling nanoflow liquid chromatography for small molecules. The resulting approach was extensively optimized and benchmarked to provide in-depth molecular coverage, along with improved chromatographic separation, sensitivity, and reliability as compared with existing methods. The cost-benefit ratio of PANAMA is substantial because the platform bypasses the need for specialized instrumentation stemming from incompatible procedures. Lin et al. develop and benchmark a unified nanoflow tandem mass spectrometry workflow enabling