Top-down proteomics: challenges, innovations, and applications in basic and clinical research

Abstract

Introduction- A better understanding of the underlying molecular mechanism of diseases is critical for developing more effective diagnostic tools and therapeutics toward precision medicine. However, many challenges remain to unravel the complex nature of diseases. Areas covered- Changes in protein isoform expression and post-translation modifications (PTMs) have gained recognition for their role in underlying disease mechanisms. Top-down mass spectrometry (MS)-based proteomics is increasingly recognized as an important method for the comprehensive characterization of proteoforms that arise from alternative splicing events and/or PTMs for basic and clinical research. Here, we review the challenges, technological innovations, and recent studies that utilize top-down proteomics to elucidate changes in the proteome with an emphasis on its use to study heart diseases. Expert opinion- Proteoform-resolved information can substantially contribute to the understanding of the molecular mechanisms underlying various diseases and for the identification of novel proteoform targets for better therapeutic development . Despite the challenges of sequencing intact proteins, top-down proteomics has enabled a wealth of information regarding protein isoform switching and changes in PTMs. Continuous developments in sample preparation, intact protein separation, and instrumentation for top-down MS have broadened its capabilities to characterize proteoforms from a range of samples on an increasingly global scale.

Keywords: Heart Diseases; Mass Spectrometry; Post-translational Modifications; Proteoforms; Top-down Proteomics.

Figure 1.

Illustration of the different components of top-down proteomics highlighting the major areas of recent development.

Figure 2.

a, Scheme illustrating photocleavable surfactant (Azo)-enabled membrane proteomics. Proteins from cardiac tissue were extracted using HEPES buffer (2x) to deplete the cytosolic proteins, followed by an extraction using 0.5% Azo. The surfactant was rapid degradation (into 4-hexylphenol, 4-hexylbenzene, nitrogen, and hydrogen sulfate under UV irradiation) of the surfactant with UV irradiation (λ_max = 305 nm) and buffer exchange before LC-MS analysis for proteoform characterization. Note that the molecules are not drawn to scale. b, Subcellular location of proteins identified in cardiac tissue in the Azo-aided top-down proteomics. c, Interactome map of the identified proteins with Azo that belong to the electron transport chain d-e, MS, and tandem MS analysis of representative membrane proteins. Phospholamban (PLN) and palmitoylated PLN (palmPLN) with palmitoylation localized at cysteine 36 residue (d), and receptor expression-enhancing protein 5 (e). The sequences below the spectra represent the fragmentation maps with sequence coverage and PTM localization based on online RPLC–MS/MS analysis. The regions representing the transmembrane domains are highlighted by blue shading. The dot represents +16 Da. Figure adapted from reference [31]. Copyright 2019 Springer Nature.

Figure 3.. Nanoproteomics enables proteoform-resolved analysis of low-abundance cardiac troponin I in human serum.

a, Silanization of Fe₃O₄ NPs using an allene carboxamide-based organosilane monomer (BAPTES) for cysteine thiol-specific bioconjugation. The rationally designed NPs 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. The photograph shows functionalized NPs in a biphasic mixture of dichloromethane (CH₂Cl₂) and water (H₂O), comparing the solvent compatibility of the NP-BAPTES and the NP-Pep. b, 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. c-d, 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, high-affinity peptide-functionalized agarose (Agarose-Pep), and antibody (mAb M46) functionalized with agarose (Agarose-mAb). e, 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), post-mortem 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. Figure adapted from reference [68]. Copyright 2020 Springer Nature.

Figure 4.

Top-down proteomics reveals a common proteoform profile in patients with hypertrophic cardiomyopathy (HCM). Coordinated decrease in cTnI and ENH2 phosphorylation in HCM tissues. Representative deconvoluted mass spectra for (A) cTnI and (B) ENH2 from donor hearts (black) and HCM tissues (red). Mono- and bis-phosphorylation are denoted by red p and pp, respectively. (C) Total protein phosphorylation (Ptot) calculated by mol Pi/mol protein for cTnI and ENH2 in Ctrl (n = 16) and HCM (n = 16). Horizontal bars represent the mean of the group and error bars represent SEM in gray for Ctrl and black for HCM. Groups were considered significantly different at P < 0.05. (D) Localization of cTnI phosphorylation to Ser22/23 and (E) localization of ENH2 phosphorylation to Ser118 by ECD. (F) Linear correlation between cTnI phosphorylation and ENH2 phosphorylation (R2 = 0.9276). Figure from reference [149]. Copyright 2020 National Academy of Sciences.

Figure 5.

A comprehensive proteomics platform for the assessment of human pluripotent stem cell (hPSC)-cardiomyocyte (CM) maturation. A, Experimental scheme for integrated top-down and bottom-up proteomics analyses of hPSC-cardiomyocytes from early- and late-stage 2-dimensional monolayer culture and 2-dimensional (3D) engineered cardiac tissue (ECT). B, Top-down liquid chromatography (LC)-mass spectrometry (MS)–based proteomics for concurrent quantification of contractile protein isoform expression and post-translational modification (PTMs). Gray and red font represent fetal and adult isoforms, respectively, in the ventricular cardiomyocytes. C, Bottom-up label-free quantitative proteomics for determining global differential regulation of proteins. CM indicates cardiomyocyte; cMyBP-C, cardiac myosin–binding protein C; cTnI, cardiac troponin I; cTnT(f)/(a), cardiac troponin T fetal/adult isoform; cα-actin, cardiac α-actin; MHC, myosin heavy chain; MLC1a, myosin light chain 1 atrial isoform; MLC1v, myosin light chain 1 ventricular isoform; MLC2a, myosin light chain 2 atrial isoform; MLC2v, myosin light chain 2 ventricular isoform; ssTnI, slow skeletal troponin I; sα-actin, skeletal α-actin; TnC, troponin C; TnI, troponin I; TnT, troponin T; and Tpm, tropomyosin. Figure from reference [151]. Copyright © 2019, Wolters Kluwer Health

Figure 6.

LC-MS profiling of myofilament proteins detected in different types of rat muscle. LC-MS of labeled myofilament protein base peak chromatograms (BPCs) of seven different rat skeletal muscles, VL, VM, VI, RF, SOL, GAS, and PLN. Reprinted (adapted) with permission from reference [154]. Melby JA, Jin Y, Lin Z et al. Top-Down Proteomics Reveals Myofilament Proteoform Heterogeneity among Various Rat Skeletal Muscle Tissues. Figure from reference [154] Journal of Proteome Research, 19(1), 446–454 (2020). Copyright 2019 American Chemical Society.