Supercharging-enhanced nDIA-MS enables global profiling of drug-induced proteome solubility shifts.

Mass spectrometry (MS) is indispensable for high-throughput quantitation of protein expression. But protein function is regulated by factors beyond abundance alone. Here, we evaluate two supercharging reagents, dimethyl sulfoxide (DMSO) and m-nitrobenzyl alcohol (mNBA), in narrow-window data-independent acquisition (nDIA)-MS. DMSO markedly enhances MS signal and protein identification, whereas mNBA primarily increases peptide identifications. Optimizating nDIA-MS with 3% DMSO boosts signal intensity by up to 56%, enabling identification of ~9,600 proteins from 1 µg HeLa digest in 15 min. Using this methodology, we quantify solubility and abundance changes in 8,694 proteins across three cell lines following short-term treatment with the proteasome inhibitor MG132 and the SUMO-activating enzyme inhibitor ML-792. MG132 affects the solubility of 1,723 proteins and the abundance of 374, and ML-792 affects 1,294 and 288, respectively. The drugs elicit distinct and sometimes opposing solubility shifts; for instance, MG132 insolubilizes HSF1, ML-792 solubilizes SP100 and insolubilizes PLOR3G, and SMAD2 shows opposite responses to those two treatments. These results reveal widespread, drug-induced remodeling of the protein solubility landscape and establish solubility profiling by nDIA-MS as a broadly applicable platform for uncovering protein state transitions and cellular responses to perturbation.