Defining the Human Heart Proteoform Landscape with Top-Down Proteomics
Author | : Trisha Tucholski |
Publisher | : |
Total Pages | : 0 |
Release | : 2020 |
Genre | : |
ISBN | : |
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Functional diversity in the heart proteome is attributed to the vast array of proteoforms arising from molecular processing events such as alternative splicing and post-translational modifications (PTMs). Given the importance of isoform switching and PTMs in the regulation of heart function and dysfunction, it is important to study the heart proteome at the proteoform level in our effort to understand heart health and disease. Top-down proteomics featuring high-resolution mass spectrometry (MS) is a premier tool for studying proteoforms in biological systems. However, the high complexity and wide dynamic range of the heart proteome have precluded in-depth top-down proteomic analysis using conventional platforms. Moreover, studying large proteoforms (>60 kDa) with top-down proteomics is especially challenging due to reduced MS signal-to-noise ratio with increasing size combined with signal suppression caused by the co-elution of smaller, more abundant proteoforms. To improve coverage of the heart proteoform landscape and access large proteoforms by top-down proteomics, serial size-exclusion chromatography (sSEC) was introduced for size-based fractionation of complex protein mixtures. A two-dimensional separation platform coupling sSEC with reversed-phase chromatography and high-resolution MS expanded coverage of heart proteoforms and enabled the detection of those up to 223 kDa from heart tissue lysate (Chapter 3). Despite improved MS detection of large proteoforms, challenges associated with tandem MS (MS/MS) fragmentation and limitations in mass resolution still prevented their identification. Intact-mass analysis software relating experimental proteoform mass to a list of candidate proteoforms enabled interpretation of complex top-down proteomic data and facilitated the identification of large heart proteoforms, including the 140-kDa myosin binding protein C (Chapter 4). Fourier transform ion cyclotron resonance (FT-ICR) MS has also provided a versatile platform for characterization of proteoforms, given its high mass resolving power and mass accuracy combined with multi-faceted fragmentation capabilities (Chapter 5). sSEC fractionation paired with top-down FT-ICR MS allowed for straightforward characterization of metabolic enzymes extracted from heart tissue (Chapter 6). Finally, top-down proteomics was applied to investigate the genotype-proteoform phenotype relationship in human hypertrophic cardiomyopathy (HCM), the most common heritable heart disease (Chapter 7). We quantified contractile proteoforms in septal myectomy tissues from HCM patients and observed converging proteoform phenotypes, regardless of disease-causing mutation, suggesting that common pathways are activated during disease progression and underscoring the importance of proteoform-level analysis. Overall, the studies detailed in this dissertation have charted new territory in the heart proteoform landscape, especially for heart proteoforms with molecular weight above 60 kDa. sSEC-based top-down proteomics facilitated cataloging of heart proteoforms to aid future studies seeking to investigate proteoforms in heart health and disease (Chapter 8). Future applications of the technology detailed herein will expand on SEC-based techniques for top-down proteomics and adapt the technology to accommodate the study of protein interactomes and large proteoforms from minute samples, such as those obtained from patients with heart disease.