Precision Profiling of the Cardiovascular Post-Translationally Modified Proteome: Where There Is a Will, There Is a Way

Abstract

There is an exponential increase in biological complexity as initial gene transcripts are spliced, translated into amino acid sequence, and post-translationally modified. Each protein can exist as multiple chemical or sequence-specific proteoforms, and each has the potential to be a critical mediator of a physiological or pathophysiological signaling cascade. Here, we provide an overview of how different proteoforms come about in biological systems and how they are most commonly measured using mass spectrometry-based proteomics and bioinformatics. Our goal is to present this information at a level accessible to every scientist interested in mass spectrometry and its application to proteome profiling. We will specifically discuss recent data linking various protein post-translational modifications to cardiovascular disease and conclude with a discussion for enablement and democratization of proteomics across the cardiovascular and scientific community. The aim is to inform and inspire the readership to explore a larger breadth of proteoform, particularity post-translational modifications, related to their particular areas of expertise in cardiovascular physiology.

Keywords: cardiovascular diseases; mass spectrometry; post-translational protein modifications; proteome; proteomics.

Figure 1

Examples of PTMs, the amino acids they target and their categories.^–

Figure 2

Schematic representation of different LC-MS/MS methods, with principal steps in the workflow for PTM analysis including data acquisition, peptides identification and quantification by mass spectrometer working on DDA, DIA and target (MRM or PRM) mode.

Figure 3

Number of proteins reported to have PTMs in Eukaryota. Summary of the number of proteins modified with the indicated PTMs in Eukaryota (blue bar), the sarcomere (grey bar) or cardiac disease (orange bar) (http://www.uniprot.org). The high number of known PTM proteins is in stark contrast to the limited knowledge about their involvement in disease. PTM: Post-translational modification. Accessing the human PTM proteome from the UniProt web site (http://www.uniprot.org). A. Select taxonomy on the left, for example, ‘Eukaryota’. B. Select ‘Reviewed’ in the ‘Map to’ section on the left. C. Use the search box to specify the PTM type. D. Additional terms can be included in the search ‘and cardiac disease’.

Figure 4

Overview of PTM analysis and challenges. Schematic representation of the well-established enrichment and mapping methods for specific PTMs workflows. PTM proteins and peptides can be enriched using a single- or multiple-step strategie. The final peptide mixture is selected and further fragmented on MS-MS to obtain informative ions that allow their identification. All fragmentations methods have advantages and disadvantages. Therefore different combinations of fragmentation techniques and mass analyzers can be used in a single analysis. Subsequently, MS/MS spectra (raw data) are processed with commercially available database search tools to identify and quantify PTM residue.