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The identification results were then exported into Microsoft Excel for manual data analysis. 2.7. Gene Ontology and Pathway Analysis The identified proteins were subjected to gene ontology (GO) analysis using Software Tool for Rapid Annotation of Proteins (STRAP) v1.5.0.0 [19]. The pathway analysis was performed using Ingenuity Pathway Analysis (IPA) software v21249400 (Qiagen, Hilden, Germany). The figures generated were abstracted from IPA. 3. Results and Discussion 3.1. 1D SDS-PAGE Showed Better Protein Extraction Efficiency with SDS and DOC In order to maximize the efficiency in the protein extraction and solubilisation of the liver tumors for downstream analysis, we compared various extraction buffers commonly used in laboratories worldwide (Table 1). Of these, SDS was chosen E-64 because it is one of the most common surfactants used to assist in the solubilisation of proteins, especially membrane proteins, during protein extraction [12, 13, 20]. Urea, a chaotrope, is another commonly used protein solubilizing agent that competes with the protein's native interactions, resulting in the unfolding of the protein and its solubilization [21]. In addition, urea is also found in TUTS buffer (one of the buffers tested in our study) which was formulated for a previous study JQ1 on the subcellular localization of membrane proteins [14]. Furthermore, thiourea, a component in the 2D extraction buffer, is found to be a stronger denaturant than urea [22]. Henceforth, the 2D extraction buffer was also included in our study. DOC is an inexpensive bile salt surfactant which has been used in studies of membrane proteins [23, 24] and was included in our study due to its comparative protein extraction efficiency as SDS [23]. Lastly, RIPA is another commonly used protein extraction buffer in proteomic studies, and it contains a small percentage of both SDS and DOC. Following protein extraction from the harvested liver tumor tissue using the various extraction buffers, 1D SDS-PAGE analysis was performed to provide an initial visual indication of protein extraction efficiency. As shown in Figure 1, protein extraction using the DOC extraction buffer was potentially better than the other extraction see more buffers, as evident from the larger number of protein bands as well as higher intensity bands in the DOC-extracted protein lysate samples. Our results were comparable to a previous study conducted by Proc et al. [23], who demonstrated that both SDS and DOC were more superior denaturants than urea in terms of the greater amount of solubilized human plasma proteins. This could explain the larger number of protein bands in the liver tumor samples extracted with SDS or DOC. However, in extraction buffers like RIPA and TUTS, the concentration of SDS and DOC could be too low to obtain comparable results with those of SDS or DOC extraction buffers.