Fig. 1: Native nanoproteomics platform for the characterization of endogenous cTn complex from human heart tissue.

a Sarcomeric proteins were extracted using a high ionic strength lithium chloride (LiCl) buffer at physiological pH. The heart tissue extract (loading mixture, L) is then incubated with peptide-functionalized nanoparticles (NP-Pep). Following magnetic isolation, the nonspecifically bound proteins are removed as flow through (F). The bound protein complexes are then eluted (E) off the NPs using a native elution buffer containing L-glutamic acid, L-arginine, and imidazole. Native top-down MS (nTDMS) analysis of enriched protein complexes proceeds by either (b) native size exclusion chromatography (SEC) for online buffer exchange and rapid analysis, (c) ultrahigh-resolution Fourier transform ion cyclotron (FTICR)-tandem MS (MS/MS) analysis for probing complex heterogeneity, stoichiometry, and localization of Ca(II) ions, or (d) trapped ion mobility spectrometry (TIMS) coupled with MS (timsTOF) for structural characterization of complex-Ca(II) binding dynamics. e High-resolution mass spectra of endogenous cardiac troponin (cTn) heterotrimeric complexes enriched by NP-Pep directly from human heart tissue and analyzed by nTDMS. f Structural representation showing the cTn heterotrimeric complex. Troponin C (TnC) is depicted in green, cardiac troponin I (cTnI) in purple, cardiac troponin T (cTnT) in orange, and Ca(II) ions in yellow. PDB: 1J1E.