Fig. 1: The vast majority of anticancer drugs induce a wide range of protein damage.

a Schematic diagram of PROTEOSTAT staining for unfolded and aggregated proteins. b MDA-MB-231 cells were treated with 101 anticancer drugs for 1 h, and protein aggregation was tested by the PROTEOSTAT staining. Drugs were classified into subtypes based on P values which ranged from 0.63 to 1.86e–06. c MDA-MB-231 cells were treated with CIS or LAP for indicated time, and protein aggregation was tested by the PROTEOSTAT staining. d The working mechanisms of 101 anticancer drugs and the associated types of protein damage. e MDA-MB-231 cells were treated with indicated anticancer drugs (n = 11) for 8 h, and the total protein carbonylation was detected by western blotting. f MDA-MB-231 cells were treated with MDV or CIS for indicated time, and total protein carbonylation was detected by western blotting. g, h MDA-MB-231 cells were treated with indicated drugs for 3 h, and biotin was pulled down. Interacting proteins were stained with Coomassie brilliant blue and K48 polyUB was detected by western blotting (g). CIS-biotin and biotin-pulled down proteins were analyzed by MS. Enhanced binding proteins (red) were highlight by log₂FC > 1, P value < 0.05 and analyzed by KEGG enrichment (h). i CIS-biotin treatment was further combined with BTZ, and CIS-biotin-pulled down proteins were analyzed by MS. Enhanced binding proteins (red) were highlight by log₂FC > 1, P value < 0.05 and analyzed by KEGG enrichment. j Flow chart depicting the LiP-MS assay. MDA-MB-231 cells were treated with CIS or DMSO for 1 h, followed by treatment with proteinase K (PK), LysC, and trypsin digestion and analysis by MS. Structure changes caused by CIS treatment and the binding of CIS either increase or prevent PK digestion, respectively, leading to differential MS peptide profiles. k, l MDA-MB-231 cells were treated with CIS, LAP, or VIN for 1 h, and proteins with differential MS peptide profiles were identified (k) and analyzed by KEGG enrichment (l). m PyMOL analysis of protein steric structure showing peptides identified by Lip-MS. Molecular docking predicted a CIS-binding energy of –7.4 kcal/mol.