Figure 1. Schematic representation of TPP-based drug target
discovery using samples of rat hippocampus. Then, we extract proteins
from the hippocampus following tissue homogenization and cell lysis.
Next, the samples were treated with a range of compound concentrations.
Each concentration was treated with four serial temperatures, i.e., 37C,
47C, 57C, 67C. Then, soluble proteins were separated and tryptic
digested before Mass spectrometry. Protein identification was made using
nano LC-ESI-Thermo Q Exactive Plus Orbi-Trap MS followed by Proteome
Discoverer software. Finally, data processing and computational analysis
were performed to compare with previously identified celecoxib targets
in different databases, and to explore the possible enriched biological
annotations in the identified protein targets.
Result
The amount of soluble proteins significantly decreased with increasing
temperature (Supplementary Figure 1). The declining pattern was observed
for all the five drug concentrations; 20µM, 10 µM, 5 µM, 1 µM, 0.1 µM,
and two negative controls, i.e., water and DMSO. Finally, the protein
sample treated in 20 µM drug concentration and 67℃ was chosen for
further analysis. In fact, proteins start unfolding at high temperature
unless the binding energy of any binding partner such as a drug is high
enough [48, 49]. We used the highest temperature to avoid detecting
the weak and transient interactions among Celecoxib and the proteins.
Also, we selected the highest dosage of Celecoxib to detect all potent
drug-target interactions.