FIGURE 1.

Target identification of ALA in NCI-H460 cells using PISA analysis. (A) Schematic workflow of the PISA experiment. Living NCI-H460 cells were treated with 10 μM ALA or DMSO for 2 h. Subsequently, aliquots were heated at 10 different temperatures for 3 min followed by a lysis by three cycles of freeze-thaw using liquid nitrogen. The 10 aliquots from the same group were then combined to one sample, and the soluble protein fractions were collected by an ultra-centrifugation. After TMT labeling, the samples were analyzed by mass spectrometry. (B) PISA concept of pooling together individual samples corresponding to different temperature points and thus hardware integration of the melting curve without detailed determination of its shape, and measuring ΔS_m along with statistical p-value could be used as a difference between the integral abundances of the protein in the treated and untreated samples. (C) The chemical structure of ALA. (D) PISA analysis results after ALA treatment was plotted by the volcano plot showing the −log₁₀ (p-values) versus the log₂ (ΔS_m). The ΔS_m (fold change) and p-value were calculated from three biological replicates of ALA treatment compared with solvent control group. Data were preprocessed using MaxQuant and plotted by Prism. The original data for Prism was provided in Supplementary Table S2.