Proteomics in heart failure: top-down or bottom-up?

Abstract

The pathophysiology of heart failure (HF) is diverse, owing to multiple etiologies and aberrations in a number of cellular processes. Therefore, it is essential to understand how defects in the molecular pathways that mediate cellular responses to internal and external stressors function as a system to drive the HF phenotype. Mass spectrometry (MS)-based proteomics strategies have great potential for advancing our understanding of disease mechanisms at the systems level because proteins are the effector molecules for all cell functions and, thus, are directly responsible for determining cell phenotype. Two MS-based proteomics strategies exist: peptide-based bottom-up and protein-based top-down proteomics--each with its own unique strengths and weaknesses for interrogating the proteome. In this review, we will discuss the advantages and disadvantages of bottom-up and top-down MS for protein identification, quantification, and analysis of post-translational modifications, as well as highlight how both of these strategies have contributed to our understanding of the molecular and cellular mechanisms underlying HF. Additionally, the challenges associated with both proteomics approaches will be discussed and insights will be offered regarding the future of MS-based proteomics in HF research.

Figure 1. Schematic illustration of the difference between top-down and bottom-up proteomics

In top-down proteomics (left), protein are extracted from cell or tissue lysates, separated by either gel or LC, and directly analyzed by MS for a complete view of all proteoforms including those with PTMs and sequence variations. Subsequently, a specific proteoform can be isolated and fragmented by MS/MS to obtain sequence information, which can be used to identify the protein via database searching, and localize PTMs. In bottom-up proteomics (right), proteins extracted from cells or tissue are subjected to proteolytic digestion (often using trypsin)—either in-solution or in-gel—and the resulting peptides are separated using LC and analyzed by MS. Subsequently, the most abundant peptides are fragmented and the peptide sequence information is used to identify the proteins present in the sample as well as map their PTMs although incomplete sequence coverage can preclude PTM analysis with this method. Numerous strategies are also available for both the relative and absolute quantification of proteins/peptides using bottom-up and top-down proteomics.

Figure 2. High-resolution Fourier transform MS (FTMS) analysis of intact rat cTnI purified from Wistar-Kyoto (WKY) and SHR myocardium

(A) Representative FTMS spectrum of cTnI (M²⁵⁺) purified from age-matched WKY. Dashed arrow indicates the expected position of tris-phosphorylated cTnI (_pppcTnI) which is not observed in this spectrum. The minor proteolytic fragment, cTnI A[16–205]K, was observed. (B) FTMS spectrum of cTnI purified from SHR-HF. _pppcTnI was observed in this spectrum. Circles represent theoretical isotopic abundance distribution of the isotopomer peaks. _pcTnI, mono-phosphorylated cTnI; _ppcTnI, bis-phosphorylated cTnI. Calc’d, calculated most abundant mass; Expt’l, experimental most abundant mass. Insets represent the schematic illustration of cTnI with differential phosphorylation in WKY and SHR, respectively. Quantification of cTnI phosphorylation in WKY and SHR-HF hearts. The percentages of mono-phosphorylated cTnI components (%P_mono) in (C); bis-phosphorylated cTnI (%P _bis) in (D); and the total amount of cTnI phosphorylation (P_total, mol Pi/mol cTnI) in (E). Data points indicate average of triplicates. Average and standard error of the mean (SEM) shown in the graph. * p < 0.05; ** p < 0.001. Modified based on (14) with permissions.