Top‐down proteomics in health and disease: Challenges and opportunities

Abstract

Proteomics is essential for deciphering how molecules interact as a system and for understanding the functions of cellular systems in human disease; however, the unique characteristics of the human proteome, which include a high dynamic range of protein expression and extreme complexity due to a plethora of post-translational modifications (PTMs) and sequence variations, make such analyses challenging. An emerging “top-down” mass spectrometry (MS)-based proteomics approach, which provides a “bird’s eye” view of all proteoforms, has unique advantages for the assessment of PTMs and sequence variations. Recently, a number of studies have showcased the potential of top-down proteomics for unraveling of disease mechanisms and discovery of new biomarkers. Nevertheless, the top-down approach still faces significant challenges in terms of protein solubility, separation, and the detection of large intact proteins, as well as the under-developed data analysis tools. Consequently, new technological developments are urgently needed to advance the field of top-down proteomics. Herein, we intend to provide an overview of the recent applications of top-down proteomics in biomedical research. Moreover, we will outline the challenges and opportunities facing top-down proteomics strategies aimed at understanding and diagnosing human diseases.

MASH Suite Pro: A Comprehensive Software Tool for Top-Down Proteomics

“…Nevertheless, the bottom-up approach is suboptimal for the analysis of protein posttranslational modifications (PTMs) and sequence variants as a result of protein digestion (10). Alternatively, the protein-based top-down proteomics approach analyzes intact proteins, which provides a "bird's eye" view of all proteoforms (11), including those arising from sequence variations, alternative splicing, and diverse PTMs, making it a disruptive technology for the comprehensive analysis of proteoforms (12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24). However, the complexity of top-down high-resolution mass spectra presents a significant challenge for data analysis.…”

Top-down proteomic analysis of protein pharmaceuticals, mixtures of protein complexes, and ribosomal protein extracts by capillary zone electrophoresis-mass spectrometry

“…Consequently, these species are often underrepresented in top-down studies due to their low solubilities [83,84]. Conversely, in the bottom-up proteomics, as hydrophobic proteins are reduced and digested, a fraction of the resultant peptides are sufficiently soluble and possess adequate ionization efficiencies to enable detection of the hydrophobic protein [85]. Nevertheless, while the characterization of hydrophobic proteins via top-down proteomics remains an analytical challenge, several recent studies have reported on the use of MScompatible detergents, micelles, or lipids for the stabilization of these structures in solution and subsequent ionization in the gas phase by ESI.…”

“…For the analysis of ESI mass spectra of intact proteins, a variety of deconvolution algorithms and software packages are available from major vendors of MS instrumentation [85]. Deconvolution involves the arrangement of ESI-MS peaks into isotopic envelopes, thereby allowing for the identification of charge and molecular weight.…”

Top-down proteomics: applications, recent developments and perspectives

“…This group also characterized different phosphorylation sites and splice variants present in human and mouse cardiac tissues [151]. An overview of the recent applications of top-down proteomics in biomedical research has been provided and outlined about the challenges and opportunities facing top-down proteomics strategies aimed at understanding and diagnosing human diseases [152]. Using a top-down MS strategy, Ying Ge and co-workers linked the altered Post-translational modification of cardiac troponin-I (cTnI) to Heart Failure (HF)-associated contractile dysfunction in both animal models of HF and human clinical samples [153].…”