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 PTMs and sequence variations, make such analyses challenging. An emerging "top-down" 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 the 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 underdeveloped 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.

Keywords: Human disease; Mass spectrometry; PTMs; Proteomics; Systems biology.

Figure 1

The schematic representation of the role of top-down proteomics in understanding the mechanisms of human disease.

Figure 2. Identification of phosphorylation of cTnI as a potential biomarker for chronic heart failure

The cTnI phosphorylation level was reduced in mild hypertrophy (G2), significantly reduced in severe hypotrophy (G3), and nearly abolished in chronic heart failure (G4) compared with normal human hearts (G1). Modified based on Zhang et al. 2011[34] with permission.

Figure 3. Modification of the pilin subunit with PG by the PptB transferase

Whole-protein mass spectrometry analysis of type IV pili purified from the wild-type strain (WT) (A), from a strain harboring a S69A mutation in the pilE gene (B), from a strain harboring a S93A mutation in the pilE gene (C), and from a mutant in the NMV_885 gene (ΔpptB) (D). Adapted from Chamot-Rooke et al. 2011[39] with permission.

Figure 4. Switch in modification from glutathionylation to cysteinylation in response to environmental conditions

(A) Representative zero charge state spectra from top-down proteomics data showing switch in S-thiolation forms in the hypothetical protein YifE (STM14_4694). Fragmentation maps show the specific cysteine residue on which the switch occurs. (B) Estimated stoichiometry from intact protein mass spectra summed across the corresponding LC peak. Adapted from Ansong et al., 2013[35] with permission.