Human MC3R Stable Cell Line - HEK293

Label-free quantitative thermal proteome profiling using ion mobility-enhanced liquid chromatography-mass spectrometry provides multifunctional datasets, offering information on differential protein expression, thermal stability, and transcription factor activity. Here, researchers developed a multidimensional data analysis workflow for label-free quantitative thermal proteome profiling (TPP) experiments, integrating gene set enrichment analysis, differential protein expression analysis, and transcription factor activity inference from liquid chromatography-mass spectrometry data. They applied this workflow to study the downstream signaling processes after activation of the melanocortin 3 receptor (MC3R) by proopiomelanocortin-derived endogenous agonists (ACTH, α-MSH, and γ-MSH). The obtained information was used to map the downstream signaling pathways of MC3R and infer the transcription factors responsible for the cellular response to ligand treatment.

In the TPP experiment, researchers analyzed the remaining soluble protein components in samples of MC3R-expressing HEK293 cells after incubation with test ligands at different temperatures. They found that the thermal stability of 298 proteins changed (either stabilized or destabilized) after interaction between MC3R and its endogenous agonists. Of these, only 4 proteins were affected by all three ligands. Another 36 proteins were affected by two ligands (ACTH and α-MSH, ACTH and γ-MSH, or α-MSH and γ-MSH), and the remaining 258 proteins had their thermal stability affected by only one POMC neuropeptide (Figure 1C). Overall, ACTH, α-MSH, and γ-MSH treatment altered the thermal stability of 142, 106, and 94 proteins, respectively (Figure 1A). Among these proteins with altered thermal stability, 104 proteins also underwent phosphorylation under the action of the three ligands. However, there was no significant enrichment trend in phosphorylation among the proteins with altered thermal stability: ACTH increased the thermal stability of 19 proteins and decreased the thermal stability of 27 proteins, and these proteins also underwent phosphorylation; the corresponding numbers for α-MSH were 7 and 17; and for γ-MSH, the numbers were 8 and 21 (Figure 1B). These numbers represent approximately one-third of the total number of proteins affected by ACTH and γ-MSH, and one-quarter of the total number of proteins affected by α-MSH.

Figure 1. Overview of identified proteins and thermally stabilized or destabilized proteins. (Sandbaumhuter F A, et al., 2023)