3.2 Identification of Differentially Expressed Proteins by 4D-LFQ
We have detected a total of 3985 proteins by 4D-LFQ quantitative analysis, of which 3350 can be quantified. Compared with the CABG group, the protein quantitative AAD group exceeded 1.5 as a significant up-regulation threshold value, and less than 1/1.5 as down-regulation. 139 proteins were upregulated, and 108 proteins were downregulated (Fig. 1).
We performed subcellular localization of these differentially expressed proteins and the results showed that upregulated proteins were mainly distributed in the cytoplasm (33.09%), extracellular matrix (31.65%), nucleus (12.95%) and other parts of the cell (Fig. 2A). The majority of the downregulated proteins are mainly distributed in the extracellular matrix (25%), mitochondria (23.15%), cytoplasm (20.37%) and other parts of the cell (Fig. 2B). The percentage of upregulated proteins located in the cytoplasm, extracellular, plasma membrane was higher than that of downregulated proteins. However, a higher percentage of downregulated proteins were mitochondria and nucleus located. In addition to the above, there are 8 and 3 upregulated proteins were found to be localized in the mitochondria and peroxisome respectively.
We performed a functional enrichment analysis of Gene Ontology (GO), KEGG pathways and protein domains to further understand the differentially expressed proteins in the ascending aorta tissue of patients with AAD.
The enrichment of differentially expressed proteins in this classification or function was positively correlated with the length of the bar chart. The GO enrichment analysis demonstrated that upregulated proteins were significantly enriched in cell components and biological processes, but at a lower level of molecular function (< 4.0). The terms involved in upregulated proteins in the above three parts mainly include extracellular space, secretory granule, calcium ion binding, transmembrane signaling receptor activity, secretion by cell, leukocyte and granulocyte activation. The down-regulated proteins are significantly enriched in cellular components and biological processes, not significantly enriched in molecular functions (< 4.0), being associated with terms including extracellular matrix, myofibril, ADP-forming activity, telethonin and actin binding, aerobic respiration, actin-myosin filament sliding (Fig. 3).
KEGG is an information network that connects known molecular interactions. The enrichment analysis of KEGG pathway showed that the differentially expressed proteins were mainly enriched in 23 pathways. Among them, ECM receptor interaction, renin secretion, TGF-beta signaling pathway, platelet activation, complement and coagulation cascades and energy-producing pathways (tricarboxylic acid cycle and propanoate metabolism) are perhaps the most relevant pathways associated with AAD. The other pathways include valine, leucine and isoleucine metabolism, protein digestion and absorption, cortisol synthesis and secretion etc (Fig. 4).
The domain enrichment analysis indicated that many of the upregulated proteins contained the following domains: EGF-like calcium-binding domain;growth factor receptor cysteine-rich domain; integrin domain; complement Clr-like EGF domain (Fig. 5A). By contrast, the down regulated proteins contained leucine-rich repeat N-terminal domain; leucine-rich repeat domain, L domain-like; von Willebrand factor, type A; amine oxidase (Fig. 5B).
In order to find the nature of the differentially expressed proteins in AAD patients, we carried out GO enrichment-based clustering, KEGG pathway enrichment-based clustering, and protein domain enrichment-based clustering analysis. We divided the differentially expressed proteins into four quantitative categories according to their different expression folds: Q1 (Ratio < 0.667,108 proteins), Q2 (0.667<Ratio < 0.769,35 proteins), Q3 (1.3<Ratio < 1.5, 153proteins) and Q4 (Ratio>1.5,139proteins). Cluster analysis based on GO enrichment showed that in the biological process category, the upregulated proteins were mainly associated with secretion by cell, leukocyte activation and neutrophil mediated immunity, granulocyte activation. The downregulated proteins were mainly associated with the cellular respiration, aerobic respiration, actin-myosin filament sliding and energy metabolism process (Fig. 6A). In the molecular function, the upregulated proteins were mainly enriched in calcium ion binding, transmembrane signaling receptor activity, drug binding, ion binding and glycoprotein binding. However, most of the downregulated proteins were associated with the succinate-CoA ligase (ADP-forming) activity, telethonin binding, actin binding (Fig. 6B). The enrichment analysis of the cellular component category showed that the upregulated proteins were highly enriched in extracellular space, secretory granule and vesicle. For the downregulated proteins, we found that were associated with extracellular matrix, myofibril, contractile fiber (Fig. 6C).
The KEGG-based enrichment analysis indicated that the upregulated proteins are mainly enriched in the phagosome, hematopoietic cell lineage, dilated cardiomyopathy (DCM), hypertrophic cardiomyopathy (HCM) pathways. Conversely, ECM-receptor interaction, amino acid metabolism and energy metabolism pathways enriched in the downregulated proteins (Fig. 7A). For the differentially expressed proteins in the domain enrichment-based clustering analysis, the upregulated proteins were clustered with EGF-like domain, growth factor receptor cysteine-rich domain, integrin α and β related domain. The downregulated proteins were associated with Leucine-rich repeat N-terminal and L domain; von Willebrand factor, type A and type D domain; dimeric alpha-beta barrel; fibronectin type III (Fig. 7B).
We performed protein interaction network analysis on all groups of differential proteins, and then in order to clearly show the interaction relationship between proteins, we screened out the top 50 proteins with the closest interaction relationship and mapped the protein interaction network. This network interaction picture includes differential proteins involved in inflammation, amino acid and energy metabolism, extracellular matrix degradation, and TGF-β signaling pathways (Figure S1, Supporting Information).