Proteomics of the heart

Abstract

Mass spectrometry-based proteomics is a sophisticated identification tool specializing in portraying protein dynamics at a molecular level. Proteomics provides biologists with a snapshot of context-dependent protein and proteoform expression, structural conformations, dynamic turnover, and protein-protein interactions. Cardiac proteomics can offer a broader and deeper understanding of the molecular mechanisms that underscore cardiovascular disease, and it is foundational to the development of future therapeutic interventions. This review encapsulates the evolution, current technologies, and future perspectives of proteomic-based mass spectrometry as it applies to the study of the heart. Key technological advancements have allowed researchers to study proteomes at a single-cell level and employ robot-assisted automation systems for enhanced sample preparation techniques, and the increase in fidelity of the mass spectrometers has allowed for the unambiguous identification of numerous dynamic posttranslational modifications. Animal models of cardiovascular disease, ranging from early animal experiments to current sophisticated models of heart failure with preserved ejection fraction, have provided the tools to study a challenging organ in the laboratory. Further technological development will pave the way for the implementation of proteomics even closer within the clinical setting, allowing not only scientists but also patients to benefit from an understanding of protein interplay as it relates to cardiac disease physiology.

Keywords: cardiac; cardiovascular; mass spectrometry; posttranslational modifications; proteomics.

Graphical abstract

FIGURE 1.

Complexity of data interpretation across the integrated omics. A: the central dogma of molecular biology is represented correlating with increasing data complexity. The power of omics data is at its strongest when multiple disciplines are integrated together. Variable factors at each omic interval led to exponential increase of data complexity, and the combination of such data is the exact interface where technological advancements are needed. ciRNA, circular RNA; lnRNA, linear RNA; PTM, posttranslational modification; SNP, single-nucleotide polymorphism. B: proteomic complexities arise from multiple proteins of distinct families coming together into a multivariate functional complex, able to be regulated via external activators and inhibitors. Additionally, PTMs can modify the protein complex’s functions, further complicating the dynamic function of protein structures in situ.

FIGURE 2.

Timeline of proteomic technological advancements and key experiments. Proteomic mass spectrometry (MS) began with separation using 2-dimensional (2-D) gel electrophoresis (2DE) techniques, which was phased out in the coming decades as new approaches were developed. Highlights include the utilization of shotgun proteomics, the invention of the Orbitrap mass spectrometer, and ability to perform data-independent acquisition searches (DIA-MS). Future innovations tend to lean into increased technological fidelity for single-cell resolution proteomics and intact top-down sample acquisition (–61). DIGE, differential in-gel electrophoresis; PTM, posttranslational modification.

FIGURE 3.

Example of 2-dimensional (2-D) gel electrophoresis (2DE) workflow for rabbit cardiac tissue study on pharmacological preconditioning (PPC; A and B) and novel finding of ATP synthase β phosphorylation. A: example of a timeline of an animal experiment using preconditioning and controls during time of ischemia; numbers with apostrophes represent minutes. IPC, ischemic preconditioning. B: sample protocol for a sequential extraction of protein material for a 2-D gel analysis and downstream sample preparation for proteomic mass spectrometry (MS). ESI, electrospray ionization; IEL, isoelectric focusing; MALDI-TOF, matrix-assisted laser desorption ionization-time of flight; MS/MS, tandem MS; TFA, trifluoroacetic acid. Ci: silver stain images of the multiple spots of ATP synthase β identified based on liquid chromatography (LC)-MS identification of each spot. Cii: silver stain image of dephosphorylated tissue specifically of the region of ATP synthase showing reduction in number of spots was used to ensure that isoelectric point (pI) shift was phosphorylation and not another posttranslational modification (PTM) such as oxidation. Ciii: Western blot image of ATPase subunit β confirming identification. D: separated 2DE spots from above were cut out and run on a mass spectrometer enriching for phosphopeptides, using immobilized metal affinity chromatography (iMAC) and ESI LC-MS to ensure the unambiguous identification of each phosphorylated residue. Figure adapted from Arrell et al. (298), with permission from Circulation Research.

FIGURE 4.

General experimental workflow for cardiac proteomics sample preparation. Tissues or cells are harvested from in vitro or in vivo experiments following experimental procedures. Depending on experiment type, cardiomyocytes may be isolated from complex tissue samples. Cardiac cells and tissues are often lysed by tissue disruption, homogenization, and/or sonication. After lysis, subproteome purification steps such as organelle or posttranslational modification (PTM) enrichment may be undertaken. The protein samples are then reduced to break structural disulfide bonds and alkylated with iodoacetamide or another alkylating agent. Unfolded polypeptides are then digested with trypsin or another tryptic enzyme, creating peptides. These peptides are desalted before being separated by mass on the liquid chromatography (LC) system and ionized as the peptides enter the mass spectrometer. Data are acquired as mass-to-charge ratios (m/z), which are then deconvoluted in silico, providing a set of peptide concentrations relating to proteins present in the sample ready for downstream statistical processing and analysis. FC, fold change.

FIGURE 5.

Overview of common data analysis workflows and tools for proteomic data. Raw data files are acquired from the mass spectrometer as spectra comprising of mass-to-charge ratios (m/z). The peaks in the data are then assigned to masses and processed with a variety of software packages. Optional library generation can take place, depending on whether the platform and workflows are data-dependent acquisition (DDA) or data-independent acquisition (DIA) based. Large data arrays then undergo further statistical processing, filtering, and corrections to have normalized and comparable results. Finally, data are analyzed and may be visualized with a number of software tools to gain biological insights about the completed experiment. FC, fold change; PCA, principal component analysis.

FIGURE 6.

Experimental sample preparation techniques for subproteome fractionation and isolation. Heart tissue/cells can be processed differentially according to the desired subproteome fraction. Sequential extraction may be performed to obtain a cytosolic, myofilament, or membrane fraction. This generally requires a sequential purification of tissue/cell lysates through differential pH levels aided by sample pelleting via centrifugation to obtain the desired fraction. Neutral sample pH will separate cytosolic proteins; acidic pH will purify myofilament-related proteins; and the residual pellet will contain membrane material. Differential centrifugation, often utilizing an ultracentrifuge without altering the sample pH levels, can be used for isolation of mitochondria, nuclei, and chromatin. The desired organelle fractions will separate by mass, allowing for independent removal of desired fractions. Affinity capture and other chemical biology approaches are useful to isolate specific protein complexes and for the enrichment of posttranslational modifications (PTMs). These captures are usually performed in vitro, although several modern labeling techniques [such as azidohomoalanine (AHA) labeling and in vivo crosslinking] may be utilized within living cells. All above methods yield a cellular subproteome, allowing for an increased dynamic range and quality of mass spectrometry (MS) data.

FIGURE 7.

Diagram of possible posttranslational modifications (PTMs) and their sites of incorporation. Commonly represented PTMs are portrayed here with lines linking them to their cognate residues of installation, denoted by the 1-letter amino acid code. Color scheme of residues refers to the different residue types. Representation of several PTMs in diagrams can additionally be found, highlighting the important structural differences between polypeptide-based PTMs (ubiquitination, neddylation, etc.); oligosaccharide-based PTMs (N-linked glycosylation, etc.); and chemical modifiers as PTMs (methylation, oxidation, etc.). SUMO, small ubiquitin-related modifier.

FIGURE 8.

Common phosphopeptide enrichment and fragmentation strategies for mass spectrometry (MS)-based analysis of the phosphoproteome. A typical ATP-dependent protein phosphorylation schematic can be viewed on left with associated downstream effects. Within sample processing, a common step for phosphopeptide enrichment is immediately following tryptic digestion steps. These typically involve affinity metal capture techniques. A range of MS-based dissociation and peptide enrichment strategies can be additionally carried out in subsequent steps to increase phosphopeptide purity. LC-MS/MS, liquid chromatography-tandem MS; NTA, nitrilotriacetic acid; PTM, posttranslational modification.

FIGURE 9.

Survey of common oxidative posttranslational modifications (OxPTMs) and the amino acid residues they modify. The chemistries and names of many possible OxPTMs are portrayed here. Names of reversible modifications are listed in green; irreversible modifications are listed in red. Color coding of amino acid residues is denoted in the key and is the same as in FIGURE 7.

FIGURE 10.

Various approaches for the identification of the Ubiquitin (Ub) and Ub-like associated proteins. Ub and Ubi-like modifiers such as NEDD8 and ISG15 are posttranslational modifications (PTMs) that confer different biological outcomes for their associated proteins upon binding, as showcased at top. However, the use of a trypsin-based digest during mass spectrometry (MS) sample preparation leaves an identical diGly motif from all 3 of the aforementioned PTMs. These diGly motifs can be purified with a specific antibody; however, discerning between the 3 PTMs becomes impossible. The use of Lys-C as a protease creates a longer Ub peptide and lends the ability to subsequently use a purifying antibody against the extended Ub peptide, separating out proteins with their specific PTMs.