Figure 6.

ALARM MSPS assay analysis of non-specific compound adduction to La antigen C245. (A) Experimental workflow to identify thiol reactivity and reversible/irreversible covalent binding of compounds to C245 of the La antigen. Compounds added at 50 mM final concentration, La antigen final concentration of 30 μM, incubated for 1 h at room temperature in the dark. (B) Heat map indicating the number of samples that afforded IAD-adducted peptide FSGDLDDQTC²⁴⁵R. Three biological replicates for each compound were run in technical triplicate. Fluconazole was used as a non-reactive negative control and CPM was used as a thiol-reactive positive control, as in the ALARM NMR assay. Data on the additional peptides analyzed containing C232 can be found in the SI. ^aThe C245 peptide was not identified in one technical replicate of one of the biological replicates, but did appear as IAD-adducted in the other two technical replicates of that sample. Therefore, the biological replicate was considered positive for IAD adduction. ^bThe FSGDLDDQTC²⁴⁵R peptide was not observed in one biological replicate, likely due to low peptide abundance since expected d₂-IAD adductions were clearly observed in other biological replicates, as well as for C232-containing peptides (see Table S12).