A Unified Mass Spectrometry Workflow Integrating Bottom-Up and Native Top-Down Proteomics via Nanodroplet Enzymatic Digestion.

Bottom-up and native top-down proteomics provide complementary but traditionally disconnected views of protein composition and structure. Integrating these approaches into a single, streamlined workflow has remained challenging due to incompatible sample preparation and analytical requirements. Here we report a unified mass spectrometry platform that bridges bottom-up and native top-down analysis through nanodroplet-accelerated enzymatic digestion performed directly under native electrospray conditions. By tuning the extent of digestion within nanodroplets, intact proteins, protein assemblies, and proteolytic peptides are generated simultaneously and analyzed within a single experiment. This workflow enables simultaneous peptide-level identification and native proteoform analysis within a single mass spectrometric experiment. We demonstrate the generality of this approach using myoglobin, amphipathic β-casein isoforms, and the 800 kDa GroEL chaperonin complex, achieving rapid sequence coverage while preserving native structural information accessible by native top-down mass spectrometry. This integrated strategy provides a practical route for comprehensive proteoform and protein assembly characterization across multiple levels of structural organization.