Native nanoproteomics captures the structure and dynamics of endogenous protein complexes in human heart tissue

Native top-down mass spectrometry (nTDMS) has emerged as a powerful structural biology tool to preserve and characterize protein complexes in their native states, but significant challenges remain in the analysis of low-abundance protein complexes. Here we have developed a "native nanoproteomics" strategy for the native enrichment and subsequent nTDMS of low-abundance protein complexes. Specifically, we demonstrate the first comprehensive characterization of the structure and dynamics of cardiac troponin (cTn) complexes directly from human heart tissue. The endogenous cTn complex is effectively enriched and purified using peptide-functionalized superparamagnetic nanoparticles under non-denaturing conditions to enable the isotopic resolution of cTn complexes, revealing their complex structure and assembly. Moreover, nTDMS elucidates the stoichiometry and composition of the heterotrimeric cTn complex, localizes Ca2+ binding domains (II-IV), defines cTn-Ca2+ binding dynamics, and provides high-resolution mapping of proteoform landscape. This native nanoproteomics strategy opens a new paradigm for structural characterization of low-abundance native protein complexes.