Fig. 4: Native top-down MS (nTDMS) enables localization of endogenous Ca(II)-binding domains within TnC. | Nature Communications

Fig. 4: Native top-down MS (nTDMS) enables localization of endogenous Ca(II)-binding domains within TnC.

From: Structure and dynamics of endogenous cardiac troponin complex in human heart tissue captured by native nanoproteomics

Fig. 4

a Isolation of native TnC monomer (z = 8+) showing proteoforms with differential Ca(II) binding using ultrahigh-resolution FTICR-MS. (Inset) Isolation of TnC + 2 Ca(II) proteoform (z = 8+). Theoretical isotope distributions (red circles) are overlaid on the experimentally obtained mass spectrum to illustrate the high mass accuracy proteoform characterization. All individual ion assignments are within 16 ppm from the theoretical mass. b MS/MS characterization of TnC + 2 Ca(II) proteoform isolated from the quadrupole window centered at 2316 m/z by collisionally activated dissociation (CAD) fragmentation. c nTDMS CAD fragmentation map showing the localization of three Ca(II) ions and N-terminal acetylation in TnC monomer achieving ~63% total bond cleavage. We observed sequential binding of Ca(II) ions to TnC with Ca(II) first binding to domain III (red), then domain IV (yellow), and lastly domain II (blue). d Structural representation of the cTn complex with the experimentally defined Ca(II)-binding domains (II–IV) highlighted (domain II, blue; domain III, red; domain IV, yellow, UniprotKB annotations, gray). TnC is depicted in green, cTnI in purple, cTnT in orange, and Ca(II) ions in yellow. PDB: 1J1E. e Magnified view of TnC Ca(II)-binding in domain II showing possible coordination with amino acid residues D73 and E77. PDB: 1J1E.

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