Combining Click Chemistry-Based Proteomics With Dox-Inducible Gene Expression.

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.