Top-down Proteomics: Technology Advancements and Applications to Heart Diseases

Abstract

Introduction: Heart diseases are a leading cause of morbidity and mortality for both men and women worldwide, and impose significant economic burdens on the healthcare systems. Despite substantial effort over the last several decades, the molecular mechanisms underlying diseases of the heart remain poorly understood.

Areas covered: Altered protein post-translational modifications (PTMs) and protein isoform switching are increasingly recognized as important disease mechanisms. Top-down high-resolution mass spectrometry (MS)-based proteomics has emerged as the most powerful method for the comprehensive analysis of PTMs and protein isoforms. Here, we will review recent technology developments in the field of top-down proteomics, as well as highlight recent studies utilizing top-down proteomics to decipher the cardiac proteome for the understanding of the molecular mechanisms underlying diseases of the heart. Expert commentary: Top-down proteomics is a premier method for the global and comprehensive study of protein isoforms and their PTMs, enabling the identification of novel protein isoforms and PTMs, characterization of sequence variations, and quantification of disease-associated alterations. Despite significant challenges, continuous development of top-down proteomics technology will greatly aid the dissection of the molecular mechanisms underlying diseases of the hearts for the identification of novel biomarkers and therapeutic targets.

Keywords: Top-down mass spectrometry; heart diseases; myofilaments; post-translational modifications; quantitative analysis.

Figure 1

A. Exponential increase of proteome complexity owing to alternative splicing of mRNAs and post-translational modifications of proteins. B. Schematic depicting the basic workflow for top-down proteomics as compared with that of bottom-up proteomics. Adapted from [24] originally published in Pflügers Archiv European Journal of Physiology, with permission from Springer and authors. © Springer

Figure 2

Schematics showing the fragment ions produced by CID (A), ECD/ETD (B), and UVPD (C). Noted that the dissociation pathways delineated are not exclusive. CID usually resulted in the loss of labile PTMs (A), whereas ECD/ETD typically preserves them (B).

Figure 3

Top-down proteomics revealed a concerted reduction of protein phosphorylation in cTnI (A), RLC (B), and an unknown protein (UnK) (C), which was subsequently identified as ENH2. D. Top-down MS/MS analysis unambiguously localized the site of ENH2 phosphorylation to Ser118, and detected a previously unknown amino acid polymorphism at Cys72. This research was originally published in Mol Cell Proteomics [77]. Adapted from [77] with permission. © The American Society for Biochemistry and Molecular Biology.

Figure 4

Top-down proteomics revealed regional heterogeneity of cTnI and α-Tm proteoforms. Basal phosphorylation of cTnI is higher in left and right ventricles versus atria (A-B), and basal phosphorylation of α-Tm is higher in the atria versus ventricles (D-E). This research was originally published in J Mol Cell Cardiol [21]. Adapted from [21] with permission from Elsevier and authors. © Elsevier