2. A B C
Figure S1. LC-MS and LC-MS/MS data on [M+H]+ ion with 837 m/z formed when MDI is reacted with oxidized glutathione. Following reactivity of MDI with purified GSSG, panel A shows mass spectrometry data on ions eluting at 2.5 minutes (see Figure 1 in manuscript). Note predominance of 837 m/z ion and its 419 m/z doubly charged species. Panel B shows fragmented ions produced upon collision induced dissociation (CID) of the 837 m/z ion. Note for Panels A & B the Y-axes depict signal intensity and the X-axes depict the mass/charge ratio. Panel C depicts proposed chemical structure for the 837 m/z parent ion and the observed fragmented ions released upon CID. 2

3. A B C
Figure S2. LC-MS and LC-MS/MS data on [M+H]+ ion with 863 m/z formed when MDI is reacted with oxidized glutathione. Following reactivity of MDI with purified GSSG, Panel A shows mass spectrometry data on ions eluting at 3.9 minutes (see Figure 1 in manuscript). Note predominance of 863 m/z ion and more limited signal from the doubly charged 432 m/z species. Panel B shows fragmented ions produced upon collision induced dissociation (CID) of the 863 m/z ion. Note for Panels A & B the Y-axes depict signal intensity and the X-axes depict the mass/charge ratio. Panel C depicts proposed chemical structure for the 863 m/z parent ion and the observed fragmented ions released upon CID. 3

4. A B C * * * * * *
Figure S3. LC-MS and LC-MS/MS data on the [M+H]+ ion with a 1475 m/z formed when MDI is reacted with oxidized glutathione. Following reactivity of MDI with purified GSSG, Panel A shows mass spectrometry data on ions eluting at ~3.2 minutes (see Figure 1 in manuscript). Note predominance of the doubly charged 738 m/z species as well as lower signal from the singly charged 1475 species. Panel B shows fragmented ions produced upon collision induced dissociation (CID) of the 1475 m/z parent ion. Panel C expands the MS/MS data in the m/z range. Note the CID fragmentation pattern supports the structures proposed in Figure 2 of the manuscript, including the 863 m/z ion consistent with GSSG-MDI (shown in Figure S2), the 613 m/z species consistent with oxidized glutathione, and related ions also noted upon CID of the 863 and/or 837 m/z species described in Figs. S1 and S2 (denoted by * in Panel C). In Panels A-C, the Y-axes depict signal intensity and the X-axes depict the mass/charge ratio. 4

5. A B C * * * * *
Figure S4. LC-MS and LC-MS/MS data on the [M+H]+ ion with a 1423 m/z formed when MDI is reacted with reduced glutathione (GSH). Following reactivity of MDI with purified GSH, Panel A shows mass spectrometry data on ions eluting at ~4.3 minutes (akin to Figure 3 in manuscript). Note predominance of the doubly charged m/z species as well as lower signal from the singly charged 1423 species. Panel B shows fragmented ions produced upon collision induced dissociation (CID) of the 1423 m/z parent ion. Panel C expands the MS/MS data in the m/z range. Note the CID fragmentation pattern supports the structures proposed in Figure 3A and B of the manuscript and includes previously published (Wisnewski et al, 2016, Xenobiotica 46:726) ions corresponding to GSH-MDI as well as its CID fragments. Signature ions include the 865 m/z ion corresponding to bis(GSH)-MDI, the m/z ion corresponding to cys-gly-MDI, the 429 m/z ion corresponding to (cys-gly)-MDI, and the 403 m/z ion corresponding to (cys-gly)-MDI*. Note the 607 m/z ion in the MS/MS spectra corresponds to bis(cys-gly)-MDI and could only arise from the structures proposed in Fig. 4A and B (not 4C or D) of the manuscript. In Panels A-C, the Y-axes depict signal intensity and the X-axes depict the mass/charge ratio. 5

6. Relative Area Under Curve
Figure S5. S9-Mediated formation of GSSG-MDI and GSH-MDI with and without an NADPH regenerating system. MDI was incubated with a liver S9 fraction with (white) or without (black) an NADPH regenerating system and the amount of GSSG-MDI* (837 m/z), GSSG-MDI (863 m/z) and GSH-MDI (558 m/z) were calculated from EICs, based on the relative area under the curve of peaks with defined m/z ratios and retention times, as described in the Methods section. The amount of glutathione-MDI conjugates did not differ significantly (p > 0.05) when an NADPH regenerating system was included in the reaction. Data shown are the mean and standard error from N=4 independent experiments. 6