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

Abstract

Summary 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 the 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.

Transformative Impact of Proteomics on Cardiovascular Health and Disease

“…81,83,[106][107][108][109] In discovery proteomics, strategies include bottom-up and top-down approaches. 106 Bottom-up strategies (peptide-level identification) are based on analysis of peptide fragments derived from parent proteins.…”

“…81,83,[106][107][108][109] In discovery proteomics, strategies include bottom-up and top-down approaches. 106 Bottom-up strategies (peptide-level identification) are based on analysis of peptide fragments derived from parent proteins. In contrast, top-down proteomic analysis (protein-level identification) allows the identification of intact proteins and is the only approach that can potentially determine the full set of PTMs that are present on a single protein molecule, thereby allowing investigation of the interrelationships of substoichiometric PTMs.…”

The clinical utility of mass spectrometry based protein assays

“…Although the MS community has largely mandated the use of stable isotope labeled internal standards for protein analysis, the exact nature of the internal standard (full, tryptic, winged, concatenated) is not fixed. Furthermore, as proteins are less frequently measured using top-down techniques and most commonly measured using a surrogate peptide derived from the enzymatic digestion of the analyte protein [118,119], the choice in surrogate peptide may also contribute to inter-laboratory differences. Therefore, due to the wide variety of options available to the laboratory scientist, two assays performed on the same protein within different laboratories might have significantly different performance characteristics, which is clearly unacceptable for use in patient care.…”

Integration of Proteomics and Metabolomics in Exploring Genetic and Rare Metabolic Diseases

“…The sample is first subjected to proteolytic digestion and then analyzed by MS; thus, proteins are identified by matching the masses of proteolytic peptides or their tandem mass spectra with theoretical in silico digestions performed by bioinformatics tools. Alternatively, new emerging “top-down” approaches do not make use of proteolytic enzymes and propose to analyze intact proteins [16]. While the former approach confirms to be the most robust strategy for protein identification ensuring better separation of peptides compared with proteins [16], the latter aims to identify all the proteoforms of a protein expressed in a sample including genetic mutations, alternative splicing variants, and posttranslational modifications (PTMs) [17].…”

“…Alternatively, new emerging “top-down” approaches do not make use of proteolytic enzymes and propose to analyze intact proteins [16]. While the former approach confirms to be the most robust strategy for protein identification ensuring better separation of peptides compared with proteins [16], the latter aims to identify all the proteoforms of a protein expressed in a sample including genetic mutations, alternative splicing variants, and posttranslational modifications (PTMs) [17]. The identification of PTMs gains a crescent importance in basic and translational research due to their implication in regulating protein structure, function, and localization [18].…”