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mz formed when MDI is reacted with oxidized glutathione Following reactivity of MDI with purified GSSG panel A shows m ass spectrometry data on ions eluting at 25 minutes see Figure 1 in ID: 931435
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Slide1
Slide2Figure 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.
A
B
C
2
Slide3Figure 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.A
B
C
3
Slide4Figure 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 100-1000 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.
A
B
C
*
*
*
*
*
*
4
Slide5Figure 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 711.7 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 100-900
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 736.20 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.
*
*
*
*
*
A
B
C
5
Slide6Relative 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