Fig 2: Design and characterization of surface-functionalized nanoparticles (NPs) for capturing cTnI.a Silanization of Fe3O4 NPs using an allene carboxamide-based organosilane monomer (BAPTES) for cysteine-thiol-specific bioconjugation. b Illustration of the rationally designed NPs that are surface functionalized with a 13-mer peptide that has a high affinity for cTnI (NP-Pep) for cTnI enrichment. The 13-mer peptide possesses a C-terminal cysteine that selectively reacts with the allene carboxamide moiety on the silanized NPs. Representative TEM images of surface-functionalized NPs: Fe3O4-OA NPs (c) (inset shows the selected area electron diffraction pattern), Fe3O4-BAPTES NPs (d), and Fe3O4-Peptide NPs (e). Representative FTIR spectra (f) and TGA analysis (g) of various NPs: Fe3O4-OA NPs (black), Fe3O4-BAPTES NPs (red), and Fe3O4-peptide NPs (blue). FTIR spectra are offset along the y-axis for clarity, with arrows denoting the characteristic allene (C = C = C) vibrational modes (1971 and 1947 cm-1) corresponding to the silanized NPs. TGA analysis reveals increased organic content on NPs after peptide coupling. h Photographs of functionalized NPs in a biphasic mixture of dichloromethane (CH2Cl2) and water (H2O), comparing the solvent compatibility of the NP-BAPTES and the NP-Pep. The NP-BAPTES are dispersible in dichloromethane but the NP-Pep are stable and dispersible in water. The displayed NP-Pep and NP-BAPTES originated from the same synthetic batch. NP-BAPTES is referred to as NP-Ctrl in the subsequent figures and text. Data are representative of n = 3 independent experiments. Source data are provided as a Source Data file.

Fig 3: The dPKC-dF1Fo interaction inhibitor reduces cardiac troponin I (cTnI) release when perfused into Langendorff hearts.Experimental details were as described in the legend to Figure 4 except that during the peptide loading phase 50 nM concentrations of the scrambled sequence (inactive) control peptide or the dPKC-dF1Fo interaction inhibitor peptide were administered to hearts for 1 hr. Effluents were collected and Western blot analyses for cTnI were conducted. The left-most lane contains rat left ventricle homogenate used as a positive control for cTnI immunoreactivity. The remaining groups are described in the legend to Figure 4. Results shown are typical of the responses of 3 independent rat hearts in each peptide treatment group.

Fig 4: Nanoproteomics enables comprehensive analysis of cTnI proteoforms from human serum.a Nanoproteomics assay utilizing NP-Pep for specific enrichment of cTnI from serum and subsequent top-down MS analysis of cTnI proteoforms. cTnI is first spiked into human serum to prepare the loading mixture (L). The NPs are then incubated with the serum loading mixture, the cTnI-bound NPs are magnetically isolated, the unwanted and nonspecific proteins are removed as flow through (F). The captured cTnI is then eluted and the final elution fraction after enrichment is analyzed by top-down LC/MS. b Deconvoluted MS corresponding to cTnI proteoforms enriched from human serum. The cTnI (~10–20 ng/mL) spiked in the human serum (10 mg) were extracted from various human hearts: (i) and (ii), donor hearts; (iii) and (iv), diseased hearts with dilated cardiomyopathy; (v) and (vi), postmortem hearts. cTnI proteoforms were identified using accurate intact mass measurement, using the most abundant mass based on the amino acid sequence of entry name TNNI3_human from the UniProtKB sequence database. p phosphorylation, pp bisphosphorylation. Data are representative of n = 3 independent experiments. A summary of the enriched proteoforms from serum and their respective mass measurements by top-down MS are listed in Supplementary Table 8. Source data are provided as a Source Data file.

Fig 5: Direct enrichment of cTnI from human serum by NP-Pep with high specificity and sensitivity.Human serum with spike-in cTnI was used for the evaluation of cTnI enrichment performance using NP-Pep and the conventional agarose-mAb. a SDS-PAGE visualizing the effectiveness of cTnI enrichment from human serum using different affinity platforms. The cTnI (306 ng) was obtained from a sarcomeric protein extract from a healthy donor heart and spiked into human serum (10 mg). Loading mixture (L), flow through (F), and elution mixture after enrichment (E) were equally loaded on the gel (500 ng) using NP-Ctrl, NP-Pep, Agarose-Ctrl, Agarose-Pep, and Agarose-mAb. The NP-Pep enabled nearly complete depletion of human serum albumin (HSA), allowing for more specific enrichment of cTnI from human serum. b Normalized extracted ion chromatograms (EICs) of cTnI (black) and HSA (red) corresponding to the NP-Pep, Agarose-Pep, and Agarose-mAb shown in (a). Normalized deconvoluted mass spectra corresponding to enriched cTnI (c) and depleted HSA (d), illustrating the abundance of cTnI and HSA before and after enrichment using NP-Pep, Agarose-Pep, and Agarose-mAb corresponding to (a, b). e Normalized EICs illustrating the MS response for cTnI (3.1–0.006 ng/ml) obtained from a human heart tissue extract (n = 2 independent experiments) using a Bruker maXis II ETD. f Evaluation of the sensitivity performance of the nanoproteomics assay. MS response against cTnI concentration is shown for various NP-Pep enrichment fractions from serum mixtures spiked with cTnI (22.53–0.50 ng/mL). The deconvoluted peak intensities of ppcTnI were used for the analyses. Data are representative of n = 3 independent experiments with error bars indicating standard error of the mean. g Representative deconvoluted mass spectra illustrating the MS response for ppcTnI corresponding to the plot in (f). M. protein marker, Std. endogenous cTnI protein standard, p phosphorylation, pp bisphosphorylation, HSA human serum albumin, C-HSA cysteinylated human serum albumin, C-C-HSA doubly cysteinylated human serum albumin, NP-Ctrl unfunctionalized NP, NP-Pep high affinity peptide-functionalized NP, Agarose-Ctrl unfunctionalized agarose, Agarose-Pep high affinity peptide-functionalized agarose, Agarose-mAb antibody (mAb M46) functionalized with agarose. Source data are provided as a Source Data file.