Figure 5

Proteomic analysis of thoracic lymph from CS-exposed mice. (A) Brightfield microscopy demonstrating the thoracic duct (TD) in mice relative to other structures. (B,C) Higher magnification image of region indicated in (A) showing harvest of lymph via cannulation of TD (arrows). (D) Label free proteomic analysis of lymph after 8 weeks of CS exposure identified 114 unique proteins in lymph from CS-exposed mice and 46 unique proteins in lymph from room air mice. 334 overlapping proteins were seen in these groups, of which 151 were upregulated and 107 were downregulated in lymph from CS-exposed mice compared to room air mice. The proteomics data set are presented in Supplementary Table 1. (E) Ingenuity pathway analysis (IPA) of DIA proteomics data set presented in the Supplementary Table 1 retrieved top biochemical pathways displaying quantitative changes in the lymph of CS-exposed mice. The experimentally determined protein ratios corresponding to [CS/Room Air] from MS1 and MS2 analysis of DIA proteomics data were used to calculate the experimental fold changes by rescaling their values using a log2 transformation, such that positive values reflected fold increases (red color) while the negative values reflected fold decreases (green color). Results from 2 independent experiments with a total of n = 10 CS-exposed mice and n = 12 room air control mice are displayed in IPA using stacked bars while the percentage overlapped proteins from the experimental data set with the IPA knowledge data base for reach pathway are shown on the X axis. The probability of having a relationship between each IPA indexed biological function and the experimentally determined protein was calculated by right-tailed Fisher’s exact test with the Benjamini–Hochberg Correction. The statistical significance was set to a P-value of < 0.05. (G) Relative abundance of coagulation proteins in lymph from CS-exposed or room air control mice, as quantified by normalized average MS1 + MS2 intensities from DIA proteomics data. (H) Relative abundance of serpine proteins in lymph from CS-exposed or room air control mice, as quantified by normalized average MS1 + MS2 intensities from DIA proteomics data. Results are reported as mean ± SDV (n = 12 for room air and n = 10 for CS samples). The statistical significance corresponding to P < 0.05 was calculated in GraphPad Prism 9 (GraphPad Software, La Jolla, CA) using the multiple nonparametric Mann–Whitney t tests discovery analysis with Holm–Sidak method (alpha = 0.05).