Absolute Quantitative Targeted Monitoring of Potential Plasma Protein Biomarkers: A Pilot Study on Healthy Individuals

Abstract

Background/objectives: The development of blood tests for the early detection of individual predisposition to socially significant diseases remains a pressing issue.

Methods: In this pilot study, multiple reaction monitoring mass spectrometry (MRM-MS) with a BAK-270 assay was applied for protein concentrations analysis in blood plasma from 21 healthy volunteers of the European cohort.

Results: The levels of 138 plasma proteins were reliably and precisely quantified in no less than 50% of samples. The quantified proteins included 66 FDA-approved markers of cardiovascular diseases (CVD), and other potential biomarkers of pathologies such as cancer, diabetes mellitus, and Alzheimer's disease. The analysis of individual variations of the plasma proteins revealed significant differences between the male (11) and female (10) groups. In total, fifteen proteins had a significantly different concentration in plasma; this included four proteins that exhibited changes greater than ±1.5-fold, three proteins (RBP4, APCS, and TTR) with higher levels in males, and one (SHBG) elevated in females. The obtained results demonstrated considerable agreement with the data collected from 20 samples of a North American cohort, which were analyzed with the similar MRM assay. The most significant differences between the cohorts of the two continents were observed in the level of 42 plasma proteins (including 24 FDA markers), of which 17 proteins showed a ≥1.5-fold change, and included proteins increased in North Americans (APOB, CRTAC1, C1QB, C1QC, C9, CRP, HP, IGHG1, IGKV4-1, SERPING1, RBP4, and AZGP1), as well as those elevated in Europeans (APOF, CD5L, HBG2, SELPLG, and TNA).

Conclusions: The results suggest a different contribution of specific (patho)physiological pathways (e.g., immune system and blood coagulation) to the development of socially significant diseases in Europeans and North Americans, and they should be taken into account when refining diagnostic panels.

Keywords: MRM; biomarkers; blood; healthy volunteers; mass spectrometry; plasma; proteomics.

Figure 1

Results of data processing for 138 proteins reliably quantified in 21 plasma samples of European cohort. (A) Ranges of average protein concentrations; each sample is marked with a different color; proteins are arranged in order of increasing concentration. (B) The degree of variability in concentrations for each analyzed protein; proteins are arranged in order of increasing concentration variability; red color bars highlight FDA-approved and/or CVD markers. (C) Average % CV calculated for each FDA (upper plot) or CVD (lower plot) biomarker measured from all technical replicates.

Figure 2

Hierarchical clustering of individual plasma samples and 138 quantified proteins, using Ward’s method and based on Manhattan distance. The upper bar shows whether the samples belong to the male (blue) or female (red) group.

Figure 3

Quantified blood plasma proteins with significantly different concentrations between North American (n = 20, [9]) and European (n = 21, current study) cohorts. (A) Volcano plot: black dots indicate non-different proteins; green dots, proteins with ≥1.5-fold change; blue dots, proteins with p < 0.01; red dots, proteins with both ≥1.5-fold change and p < 0.01. (B) Protein–protein interaction (GO analysis).

Figure 3

Quantified blood plasma proteins with significantly different concentrations between North American (n = 20, [9]) and European (n = 21, current study) cohorts. (A) Volcano plot: black dots indicate non-different proteins; green dots, proteins with ≥1.5-fold change; blue dots, proteins with p < 0.01; red dots, proteins with both ≥1.5-fold change and p < 0.01. (B) Protein–protein interaction (GO analysis).