Recent Developments in Clinical Plasma Proteomics-Applied to Cardiovascular Research

Abstract

The human plasma proteome mirrors the physiological state of the cardiovascular system, a fact that has been used to analyze plasma biomarkers in routine analysis for the diagnosis and monitoring of cardiovascular diseases for decades. These biomarkers address, however, only a very limited subset of cardiovascular diseases, such as acute myocardial infarct or acute deep vein thrombosis, and clinical plasma biomarkers for the diagnosis and stratification cardiovascular diseases that are growing in incidence, such as heart failure and abdominal aortic aneurysm, do not exist and are urgently needed. The discovery of novel biomarkers in plasma has been hindered by the complexity of the human plasma proteome that again transforms into an extreme analytical complexity when it comes to the discovery of novel plasma biomarkers. This complexity is, however, addressed by recent achievements in technologies for analyzing the human plasma proteome, thereby facilitating the possibility for novel biomarker discoveries. The aims of this article is to provide an overview of the recent achievements in technologies for proteomic analysis of the human plasma proteome and their applications in cardiovascular medicine.

Keywords: affinity proteomics; cardiovascular disease; mass spectrometry-based proteomics; plasma proteomics.

Figure 1

Workflows for the three primary analytical techniques that were deployed for proteomic analysis of plasma. (A) Mass spectrometry-based methods for the analysis of plasma typically relies on either depletion or enrichment to detect low-abundant proteins. Pre-fractionation prior to proteomic analysis is an optional step that separates the peptides in one dimension, thereby dividing the sample into a smaller fraction for later analysis. The quantification of proteins for explorative biomarker discovery is performed either label-free or using isobaric labelling reagents. (B) The SomaScan assay uses SOMAmers (blue) that are synthesized with a fluorophore, photocleavable linker as well as biotin, which is used to immobilize the SOMAmers to streptavidin beads. SOMAmer are able to capture proteins (orange) from solution followed by biotinylating of the captured proteins. The photocleavable linker is destroyed using external UV light to release the SOMAmer/protein complex back into the solution. Biotin-tagged proteins are recaptured on secondary streptavidin beads. SOMAmer dissociation is caused by denaturing of the captured protein, thereby allowing the SOMAmer reagents to hybridize to complementary sequences on a microarray chip. The abundance of each protein is derived from the amount of fluorescent intensity that is detected from each fluorophore. (C) Antibodies with the specificity for the same protein (green) are brought into close enough proximity that the attached DNA can hybridize and extend in the presence of a DNA polymerase. This process forms unique DNA barcodes for each protein in the assay, which can then be amplified and used for quantification using qPCR.

Figure 2

The estimated plasma protein concentrations from the publicly available Peptide Atlas build (Human Plasma 2021-07, accessed on 1 December 2021) [24] was used to compare the number of quantifiable protein from LC-MS analysis (with and without affinity enrichment) with proteins that were available in the Olink and SomaScan assays. Note: Not all the proteins were associated with an estimated plasma protein concentration in the Peptide Atlas build. (A) Boxplot of the estimated protein concentrations for each of the analytical techniques displaying the distribution of quantifiable proteins. The whisker boundary is defined as the 10th–90th percentile. (B) Graphical representation of the diversity in the number of quantifiable proteins in relation to the estimated protein concentrations with known cardiovascular biomarkers as examples of the dynamic range in plasma. While the curves are initially similar, it is evident that the SomaScan and Olink are able to quantify far more mid-and low-abundant proteins compared to LC-MS analysis even with the application of affinity enrichment. LC-MS analysis using affinity enrichment is able to sporadically quantify proteins in the same lower range as SomaScan and Olink. Dataset references: LC-MS: [39]; LC-MS + Enrichment: [40]; Olink: [89]; SomaScan: [90].