Developing an Affinity-Based Chemical Proteomics Method to In Situ Capture Amorphous Aggregated Proteome and Profile...

Area of Science:

• Chemical Biology
• Proteomics
• Cell Biology

Background:

• Amorphous proteome aggregation in cells is linked to disease pathogenicity.
• Capturing dynamic, non-specific aggregated proteins in situ is challenging.
• Existing methods for aggregated proteome profiling lack selectivity and sensitivity.

Purpose of the Study:

• To develop a novel chemical proteomics method for capturing amorphous aggregated proteins in live stressed cells.
• To identify the proteomic contents of these aggregates using Liquid Chromatography-Mass Spectrometry/Mass Spectrometry (LC-MS/MS).
• To investigate the role of aggregated proteomes in cellular stress responses and disease.

Main Methods:

• Development of an affinity-based chemical probe (AggLink 1.0) for selective labeling of amorphous aggregated proteins.
• Utilized chaotrope-compatible ligation for effective enrichment of labeled proteins under denaturing conditions.
• Applied the AggLink method to HeLa cells under stress conditions (HSP90 or proteasome inhibition).

Main Results:

• The AggLink method demonstrated superior enrichment selectivity, detection sensitivity, and identification accuracy compared to conventional methods.
• Revealed the heterogeneity of the aggregated proteome in response to pro-folding and pro-degradation pathway inhibition.
• The probe's fluorogenicity allowed for real-time detection of aggregated proteome location and morphology within cells.

Conclusions:

• The AggLink chemical proteomics approach enables the capture and analysis of previously non-targetable amorphous aggregated proteomes.
• Provides insights into cellular strategies for managing protein aggregation, potentially uncovering synergistic cancer cell viability reduction.
• This method advances the study of protein aggregation in disease and cellular stress.