Data-Independent Acquisition Proteomics Reveals Long-Term Biomarkers in the Serum of C57BL/6J Mice Following Local High-Dose Heart Irradiation

Figure 1. Multivariate, pathway, and cardiotoxicity analyses of the significantly differentially expressed serum proteins after local heart irradiation using 0 (control), 8, or 16 Gy. The principal component analysis (PCA) performed on normalized intensities of all proteins resulted in PC1, PC2, and PC3 as follows: PC1 15.9%, PC2 15.1%, and PC3 12.3%. The control samples are represented as yellow balls, the samples exposed to 8 Gy in green cubes, and the 16 Gy treated samples in blue pyramids (A,B). A dose-dependent alteration is observed in the pathways involved in the inflammation and lipid metabolism (C). Several proteins were identified associated with different heart pathologies (D). The pathway and cardiotoxicity scores are displayed using a purple color gradient; the darker the color, the higher the scores and, thereby, statistical significance. The score is the negative log of the p-value derived from the Fisher's Exact test. By default, the rows (pathways) with the highest total scores across the set of observations are sorted to the top. The analysis was performed using Ingenuity Pathway Analysis (IPA) (Qiagen Inc.,…

Figure 2. Graphical representation of cholesterol-associated networks based on radiation-induced alterations in the serum proteome. The protein clusters are shown at 8 Gy (A) and 16 Gy (B). The upregulated proteins are marked in red and the downregulated in green. The nodes represent proteins connected with arrows; the solid arrows represent direct interactions and the dotted arrows indirect interactions. The cholesterol nodes are marked inside red circles and boxes. The IPA codes and corresponding full protein names are shown in Table 1 and in Supplementary Table 1. The analysis was performed using the Ingenuity Pathway Analysis (IPA) (https://www.qiagenbioinformatics.com/products/ingenuity-pathway-analysis).

Figure 3. Predicted upstream regulators of the deregulated serum proteins. Predicted upstream regulators are displayed using a purple color gradient where the intensity of the purple color corresponds to statistical significance (the deeper the color, the higher the significance). The score is the negative log of the p-value derived from Fisher's exact test. By default, the rows (upstream regulators) with the highest total scores across the set of observations are sorted to the top (A). The predicted upstream regulators and their activity status at 16 Gy are shown: TGF-β (B), IL-6 (C), PPARα (D), and STAT3 (E). The orange and the blue color of the nodes indicate activation and deactivation, respectively; the solid arrows represent direct interactions and the dotted arrows indirect interactions. The deregulated proteins forming the wheel around the nodes are marked in red (upregulation) and green (downregulation). The IPA codes and corresponding full protein names are shown in Table 1 and in Supplementary Table 1. The analysis was performed using Ingenuity Pathway Analysis (IPA) (https://www.qiagenbioinformatics.com/products/ingenuity-pathway-analysis).

Figure 4. The ELISA analysis of serum cytokines. The level of cytokines was measured in 100 μg of serum in mice following 8 or 16 Gy local heart radiation using ELISA (t-test; *p < 0.05, mean with SEM, n = 5).

Figure 5. The ELISA analysis of serum lipid levels. The levels of free fatty acid (FFA), triglyceride (TG), total cholesterol, HDL, LDL, and oxidized low-density lipoprotein (oxLDL) were measured in 100 μg of serum of mice at 8- or 16-Gy local heart irradiation using ELISA (t-test; *p < 0.05, mean with SEM, n = 5).