Department of Pharmaceutical Biosciences, Uppsala University, 751 24 Uppsala, Sweden.
Science for Life Laboratory, Spatial Mass Spectrometry, Uppsala University, 751 24 Uppsala, Sweden.
Anal Chem. 2023 Oct 17;95(41):15400-15408. doi: 10.1021/acs.analchem.3c03643. Epub 2023 Oct 7.
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.
采用无标记定量离子淌度增强 LC-MS 的热蛋白质组分析提供了多功能数据集，提供了有关蛋白质差异表达、热稳定性和转录因子活性的信息。我们开发了一种多维数据分析工作流程，用于无标记定量热蛋白质组分析（TPP）实验，该工作流程结合了基因集富集分析、差异蛋白质表达分析以及从 LC-MS 数据推断转录因子活性的方面。我们将其应用于研究内源性激动剂（源自前阿黑皮素原激素：ACTH、α-MSH 和 γ-MSH）激活黑皮质素 3 受体（MC3R）下游的信号转导过程。获得的信息用于绘制 MC3R 下游的信号通路，并推断负责细胞对配体处理反应的转录因子。使用我们的工作流程，我们鉴定了差异表达的蛋白质并研究了它们的热稳定性。我们发现共 298 种蛋白质的热稳定性因 MC3R 激活而改变。其中，几种蛋白质是转录因子，表明它们是参与 MC3R 信号级联的下游靶标调节剂。我们发现转录因子 CCAR2、DDX21、HMGB2、SRSF7 和 TET2 的热稳定性发生改变。这些在 MC3R 信号级联中发现的明显靶转录因子在免疫反应中发挥重要作用。此外，我们推断了我们在数据集中鉴定的转录因子的活性。这是使用我们用无标记定量 LC-MS 获得的差异表达数据，通过贝叶斯统计来完成的。我们通过观察到的磷酸化肽丰度在我们的生物信息学管道中验证了推断的转录因子活性，突出了翻译后修饰在转录因子调控中的重要性。我们的多维数据分析工作流程允许对 MC3R 激活下游的信号转导过程进行全面表征。它提供了有关蛋白质差异表达、热稳定性和关键转录因子活性的深入了解。本研究中生成的所有蛋白质组学数据都可在 DOI：10.6019/PXD039945 上公开获取。
