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

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.

采用离子迁移增强液相色谱-质谱联用（ion-mobility-enhanced LC–MS）的无标记定量（label-free quantitation）热蛋白质组分析（thermal proteome profiling, TPP）可产出多功能数据集，能够提供蛋白质差异表达、热稳定性以及转录因子活性相关信息。我们针对无标记定量热蛋白质组分析实验开发了一套多维数据分析流程，该流程整合了基因集富集分析、蛋白质差异表达分析以及从液相色谱-质谱联用数据中推断转录因子活性的相关模块。我们将该流程应用于探究由前阿黑皮素原（proopiomelanocortin prohormone）衍生的内源性激动剂（促肾上腺皮质激素ACTH、α-促黑素细胞激素α-MSH与γ-促黑素细胞激素γ-MSH）激活黑皮质素3受体（melanocortin 3 receptor, MC3R）后的下游信号传导过程。所得信息被用于绘制MC3R下游信号通路图谱，并推导配体处理后细胞应答所涉及的转录因子。借助本数据分析流程，我们鉴定出了差异表达蛋白质，并对其热稳定性进行了分析。我们共发现298种因MC3R激活而发生热稳定性改变的蛋白质，其中部分为转录因子，表明它们是参与MC3R信号级联反应的下游靶标调控因子。我们发现转录因子CCAR2、DDX21、HMGB2、SRSF7以及TET2的热稳定性发生了改变。这些位于MC3R信号级联反应中的潜在靶标转录因子在免疫应答中发挥着重要作用。此外，我们还对本数据集所鉴定的转录因子活性进行了推断，该推断基于无标记定量液相色谱-质谱联用获得的差异表达数据，采用贝叶斯统计（Bayesian statistics）方法完成。我们通过观测到的磷酸化肽段（phosphorylated peptide）丰度，在生物信息学流程中对推断得到的转录因子活性进行了验证，凸显了翻译后修饰（post-translational modifications）在转录因子调控中的重要性。本多维数据分析流程可实现对MC3R激活后下游信号传导过程的全面表征，能够为蛋白质差异表达、热稳定性以及关键转录因子活性的研究提供新视角。本研究产生的所有蛋白质组学数据均可在DOI: 10.6019/PXD039945获取。