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Combining Click Chemistry-Based Proteomics With Dox-Inducible Gene Expression
J Gebert1, M Schnölzer2, U Warnken2
1Institute of Pathology, University Hospital Heidelberg, Heidelberg, Germany; Cancer Early Detection, German Cancer Research Center (DKFZ), Heidelberg, Germany.
Abstract:
Inactivating mutations in single genes can trigger, prevent, promote, or alleviate diseases. Identifying such disease-related genes is a main pillar of medical research. Since proteins play a crucial role in mediating these effects, their impact on the diseased cells' proteome including posttranslational modifications has to be elucidated for a detailed understanding of the role of these genes in the disease process. In complex disorders, like cancer, several genes contribute to the disease process, thereby hampering the assignment of a proteomic change to the corresponding causative gene. To enable comprehensive screening for the impact of inactivation of a gene, e.g., loss of a tumor suppressor in cancer, on the cellular proteome, we present a strategy based on combination of three technologies that is recombinase-mediated cassette exchange, click chemistry, and mass spectrometry. The methodology is exemplified by the analysis of the proteomic changes induced by the loss of a tumor suppressor gene in colorectal cancer cells. To demonstrate the applicability to screen for posttranslational modification changes, we also describe the analysis of protein glycosylation changes caused by the tumor suppressor inactivation. In principle, this strategy can be applied to analyze the effects of any gene of interest on protein expression as well as posttranslational modification by glycosylation. Moreover adaptation of the strategy to an appropriate cell culture model has the potential for application on a broad range of diseases where the disease-promoting mutations have been identified.
Insights
Researchers developed a new strategy to analyze how gene inactivation affects cellular proteomes and posttranslational modifications. This method aids in understanding gene roles in diseases like cancer by combining recombinase-mediated cassette exchange, click chemistry, and mass spectrometry.
Area of Science:
- Molecular Biology
- Proteomics
- Genetics
Background:
- Identifying disease-related genes is crucial for medical research.
- Proteins and their posttranslational modifications are key mediators of gene function in disease.
- Complex disorders like cancer involve multiple genes, complicating the link between proteomic changes and specific genes.
Purpose of the Study:
- To present a novel strategy for comprehensive screening of gene inactivation effects on the cellular proteome.
- To enable detailed analysis of protein expression and posttranslational modifications, such as glycosylation, upon gene loss.
- To facilitate understanding of gene roles in disease pathogenesis, particularly in complex disorders.
Main Methods:
- Integration of recombinase-mediated cassette exchange (RMCE) for gene manipulation.
- Application of click chemistry for molecular labeling and detection.
- Utilization of mass spectrometry for high-throughput proteomic analysis.
- Exemplification using colorectal cancer cells with tumor suppressor gene loss.
Main Results:
- Demonstrated a strategy to analyze proteomic changes induced by tumor suppressor gene inactivation in colorectal cancer cells.
- Showcased the ability to screen for posttranslational modification changes, specifically protein glycosylation, following gene inactivation.
- Validated the combined approach for assessing gene impact on protein expression and glycosylation.
Conclusions:
- The presented strategy effectively screens for proteomic and posttranslational modification changes upon gene inactivation.
- This methodology is adaptable for studying any gene of interest and its effects on protein expression and glycosylation.
- The approach holds potential for broad application in diseases with identified causative mutations, aiding in understanding disease mechanisms.
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