Supercharging-Enhanced nDIA-MS Enables Global Profiling of Drug-Induced Proteome Solubility Shifts (co-solvent)

Mass spectrometry (MS)-based proteomics has become indispensable for high-throughput quantitation of protein expression. However, protein function is regulated by a vari-ety of factors beyond abundance alone. Here, we optimized narrow-window, data-independent acquisition (nDIA) MS with 3% DMSO as a supercharging co-solvent, boosting signal intensity by up to 56% and enabling identification of ~9,600 proteins from 1 µg of HeLa digest with a 15 min gradient. Using this methodology, we quantified solubility and abundance changes in 8,694 proteins across three cell lines following short-term treatment with the proteasome in-hibitor MG132 and the SUMO-activating enzyme inhibitor ML-792. MG132 affected the solubil-ity of 1,723 proteins and the abundance of 374, and ML-792 impacted 1,294 and 288, respec-tively. The drugs elicited distinct and sometimes opposing solubility shifts; For instance, MG132 insolubilized HSF1, ML-792 solubilized SP100 and insolubilized PLOR3G, and SMAD2 showed opposite responses to those two treatments. These results reveal wide-spread, drug-induced remodeling of the protein solubility landscape and establish solubility profiling by nDIA-MS as a robust and broadly applicable platform for uncovering protein state transitions and cellular responses to perturbation.