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:

• Top-down Proteomics
• Mass Spectrometry
• Post-translational Modifications
• Proteoforms
• Heart Diseases

Article highlights

• Top-down proteomics represents a powerful technology for comprehensively characterizing proteoforms for the discovery of novel biomarkers and the elucidation of disease mechanisms.

• Recently, significant advancements have been made in the sample preparation for improved protein solubilization, sample clean-up, and protein/proteoform enrichment.

• The development of intact protein separations has advanced the scope of top-down proteomics for global proteoform characterization.

• Advancements in mass spectrometry instrumentation and fragmentation have pushed the boundaries of top-down proteomics to effectively analyze large proteoforms (>2 MDa) and complex mixtures of proteoforms.

• A number of exciting applications of top-down proteomics have been demonstrated for biomedical research for better understanding and diagnosing of diseases, particularly for cardiovascular research.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Declaration of interest

The University of Wisconsin–Madison has filed a provisional patent application P180335US01, US serial number 62/682,027 (June, 7 2018) on the basis of this work. Y.Ge and K.A. Brown are named as inventors on the provisional patent application. The University of Wisconsin—Madison has filed a provisional patent application serial No. 62/949,869 (18 December 2019) on the basis of this work. Y. Ge and D.S. Roberts are named as the inventors on the provisional patent application. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

Reviewer disclosures

Peer reviewers on this manuscript have no relevant financial or other relationships to disclose.

Additional information

Funding

This paper was supported by the U.S. Foundation for the National Institutes of Health: R01 HL096971, R01 GM117058, GM125085, and HL109810 (to Y. Ge). K.A. Brown would like to acknowledge Cardiovascular Research Center Training Program in Translational Cardiovascular Science, T32 HL007936-19 and Vascular Surgery Research Training Program grant T32HL110853.  J.A. Melby would like to thank Cardiovascular Research Center Training Program in Translational Cardiovascular Science, T32 HL007936-20.