Serum-free media is necessary to avoid small molecule scavenging and is a standard incubation condition for cellular exposure to acetochlor . Indeed, the use of complete media ablates HSR activation by acetochlor. Surprisingly, Flag DNAJB8 H31Q sensitizes the cells to activate the HSR in response to acetochlor treatment, possibly due to its direct binding of misfolded client proteins . This sensitization was not previously observed for HSR induction by arsenite or by cadmium . 1 mM propachlor and 1 mM alachlor in serum-free media for 30 minutes were chosen to be consistent with the acetochlor conditions despite neither herbicide upregulating HSPA1A (Figure ). Using the same conditions for each herbicide treatment allows us to relate effects on the proteome to structural characteristics rather than to incubation time and concentration. Even in the absence of adequate global proteostatic stress for HSF1 activation, propachlor and alachlor could still induce misfolding of specific protein targets with consequent cellular toxicity . Consistent with this possibility, we found that 30 min. incubation with 1 mM propachlor inhibits viability of HEK293T cells by resazurin assay (Figure ). We utilized the Hsp40 affinity approach (Scheme 1) to profile for chloroacetanilidedependent misfolded proteomes. Flag DNAJB8 H31Q was transiently overexpressed in HEK293T cells, followed by 30 min of chloroacetanilide herbicide treatment, and immediate Flag immunoprecipitation from cellular lysate. Co-immunoprecipitated proteins were labeled by TMT isobaric tagging and then identified and quantified by LC/LC-MS/MS. Each herbicide treatment (acetochlor, alachlor, and propachlor) was performed over 24 biological replicates (12 treated and 12 controls) and analyzed by four 6-plex TMT runs. The overall protein distribution in eluates, as analyzed by SDS-PAGE separation followed by silver staining, does not show gross differences between the immunoprecipitates, as expected from such a short treatment (Figure ). reactive to a general iodoacetamide probe or any of 128 chloroacetamide probes in Kuljanin et al. . Volcano plots can be found in Figures .