DrPISA: Deep eutectic solvent-assisted reverse proteome-integrated solubility alteration for high-sensitivity drug...

Area of Science:

• Chemical proteomics
• Proteomics
• Drug discovery

Background:

• Traditional thermal stability-based proteomic methods like TPP and PISA primarily analyze soluble proteins, neglecting heat-induced aggregates.
• Conventional denaturants have limitations in solubilizing aggregated proteins and are not always compatible with proteomic workflows.
• There is a need for improved methods to access and analyze the insoluble proteome for comprehensive target identification.

Purpose of the Study:

• To develop a novel deep eutectic solvent (DES)-assisted reverse PISA (DrPISA) strategy.
• To enhance the accessibility of the insoluble proteome and improve detection sensitivity for thermal stability alterations.
• To overcome limitations of conventional denaturants in solubilizing heat-aggregated proteins.

Main Methods:

• Systematic evaluation of 65 DES formulations to identify optimal solubilization reagents for heat-aggregated proteomes.
• Integration of the optimal DES (DES-48) into a reverse PISA workflow.
• Application of a simplified six-temperature dimethyl labeling workflow for quantitative mass spectrometry.
• Utilizing DrPISA for target deconvolution and identification of protein interactions.

Main Results:

• DES-48 demonstrated superior solubilization of heat-aggregated proteomes compared to GuHCl and urea, identifying significantly more proteins.
• DrPISA enabled sensitive detection of early-stage protein aggregation events missed by soluble-focused assays.
• The method reproducibly identified known targets, increased kinase coverage, and detected subtle kinase responses.
• A simplified labeling workflow reduced reagent consumption and MS acquisition time by over 50%.
• Application to celastrol identified LULL1 as a previously under-recognized interacting protein.

Conclusions:

• DrPISA extends thermal profiling to insoluble protein fractions, outperforming conventional methods.
• The developed DES-48 reagent enhances the recovery and quantification of heat-aggregated proteins.
• DrPISA offers a scalable and sensitive approach for target deconvolution and broadens the scope of chemical proteomics.
• This strategy provides a practical complementary approach for expanding the landscape of discoverable drug-protein interactions.