Proteomics analysis of protein phosphorylation, also known as phosphoproteomics, is a powerful tool for understanding cellular signaling pathways and regulation. By identifying the phosphorylated proteins in a given sample, researchers can gain insights into the activity and interactions of proteins involved in various biological processes, such as cell division, growth, and differentiation. The application of phosphoproteomics has led to the identification of novel drug targets and biomarkers for various diseases, including cancer and neurological disorders.
Case 1. Phosphoproteomic Profiling of Synovial Sarcoma: ALK and MET Identified as Targets (1)
The authors of this article used phosphoproteomics to study the mechanism of sarcomas, a rare and diverse type of malignant tumor that commonly affects bone and connective tissue, and to find new therapeutic targets.
They selected 20 sarcoma cell lines for phosphoproteomic analysis and identified a total of 1,090 tyrosine phosphorylation peptides/sites from 654 proteins using phosphoproteomics. They then used the expression levels of these phosphorylated peptides to perform hierarchical clustering analysis.
The study compared the quantitative results of modification profiles among different cell lines, and identified the protein ALK with significant expression differences in modifications for preliminary validation and research.
Through high-throughput analysis of phosphoproteomics, the study screened proteins and sites with changes in modification levels, found key regulatory mechanisms, and confirmed the effectiveness of intervention therapy.
Case 2. Phosphoproteomics Reveals In Vivo Brain GPCR Signaling (2)
G protein-coupled receptors (GPCRs) superfamily is the main drug target for neurologic disorders. However, even stimulation of a single GPCR often activates many parallel signaling pathways, which leads to various side effects of GPCR-targeted agonists.
In this study, phosphoproteomic profiling was used to investigate the changes in protein phosphorylation modifications in response to different KOR agonists treatment, at different time points, and in different brain regions. Although a single phosphoproteomic technique was used, the experiment set multiple factors and obtained high-throughput phosphorylation modification information. Therefore, analyzing experimental data layer by layer became a critical step.
Firstly, the researchers analyzed the differences in phosphorylation modifications in various brain regions in the unstimulated state, and found region-specific features. Secondly, they analyzed the changes in phosphorylation modifications in different brain regions after agonist stimulation, and found region-specific and time-dependent features. Based on the phosphorylation disturbance, the researchers chose to perform in-depth analysis in the striatum 5 minutes after U-50,488H agonist injection. Subsequently, the synaptic signaling pathway in the striatum was analyzed in-depth, and the dephosphorylation phenomenon caused by agonist treatment was discovered. Using a phosphatase inhibitor as verification, the researchers confirmed the important role of phosphatases in this phenomenon. Finally, the researchers focused on the mTOR signaling pathway modification differences in the striatum, and combined with mTOR function and subsequent verification, they discovered the mechanism of aversive side effects caused by U-50,488H - mTOR signaling pathway.
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References
- Fleuren, Emmy DG, et al. "Phosphoproteomic profiling reveals ALK and MET as novel actionable targets across synovial sarcoma subtypes." Cancer Research 77.16 (2017): 4279-4292.
- Liu, Jeffrey J., et al. "In vivo brain GPCR signaling elucidated by phosphoproteomics." Science 360.6395 (2018): eaao4927.
