Label-Free Quantitative Thermal Proteome Profiling Reveals Target Transcription Factors with Activities Modulated by MC3R Signaling

Abstract

Thermal proteome profiling with label-free quantitation using ion-mobility-enhanced LC–MS offers versatile data sets, providing information on protein differential expression, thermal stability, and the activities of transcription factors. We developed a multidimensional data analysis workflow for label-free quantitative thermal proteome profiling (TPP) experiments that incorporates the aspects of gene set enrichment analysis, differential protein expression analysis, and inference of transcription factor activities from LC–MS data. We applied it to study the signaling processes downstream of melanocortin 3 receptor (MC3R) activation by endogenous agonists derived from the proopiomelanocortin prohormone: ACTH, α-MSH, and γ-MSH. The obtained information was used to map signaling pathways downstream of MC3R and to deduce transcription factors responsible for cellular response to ligand treatment. Using our workflow, we identified differentially expressed proteins and investigated their thermal stability. We found in total 298 proteins with altered thermal stability, resulting from MC3R activation. Out of these, several proteins were transcription factors, indicating them as being downstream target regulators that take part in the MC3R signaling cascade. We found transcription factors CCAR2, DDX21, HMGB2, SRSF7, and TET2 to have altered thermal stability. These apparent target transcription factors within the MC3R signaling cascade play important roles in immune responses. Additionally, we inferred the activities of the transcription factors identified in our data set. This was done with Bayesian statistics using the differential expression data we obtained with label-free quantitative LC–MS. The inferred transcription factor activities were validated in our bioinformatic pipeline by the phosphorylated peptide abundances that we observed, highlighting the importance of post-translational modifications in transcription factor regulation. Our multidimensional data analysis workflow allows for a comprehensive characterization of the signaling processes downstream of MC3R activation. It provides insights into protein differential expression, thermal stability, and activities of key transcription factors. All proteomic data generated in this study are publicly available at DOI: 10.6019/PXD039945.

Stability-based approaches in chemoproteomics

“…DIA quantitation is being increasingly adopted for measuring protein solubility in thermal shift assays (Refs 51 , 52 ). As outlined in our previous work, different label-free DIA approaches within a 1D-TPP workflow perform comparably to TMT-DDA (Ref.…”

“…Additionally, it presents a simplified sample preparation workflow as there is no labelling or fractionation, and reduced experimental costs in studies with large sample numbers (e.g. TMT reagents required for 2D-TPP) (Refs 51 , 52 ). Furthermore, iTSA was recently coupled with a fully automated sample preparation platform (autoSISPROT) and DIA quantification to screen 20 kinase inhibitors in a fully automated manner (Ref.…”