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Supplemental Figure 1: Chemical Structures of S-cysteinylated (15963.38 Da) and S-thiolated (16149.44 Da) of SOD1 (15844.44 Da).

Supplemental Figure 2: Mechanisms of S-thiolation. Top: Thiolate-disulfide exchange can occur anaerobically with cysteine thiolate and any disulfide-containing small molecule. It is illustrated (top) for the reaction of cystine with SOD1, which yields Cys-bound-SOD1 (+119 Da) and free cysteine. Bottom: Disulfide bond formation starting from only sulfhydryl-containing compounds requires enzyme- or small molecule (e.g. oxygen)-mediated oxidation. It is illustrated (bottom) for the reaction of glutathione with SOD1.

Supplemental Figure 3: Thiol scavengers, iodoacetamide, iodoacetic acid, and MMTS, did not reverse cysteinylation of SOD1. Recombinant SOD1 was cysteinylated with 40-fold molar excess of L-cysteine. SOD1 was then incubated in the presence or absence of the thiol scavengers (iodoacetamide, iodoacetic acid, and/or MMTS) and homogenized using the same protocol that was used to homogenize tissue samples. After homogenization, the supernatants were incubated overnight (thiol scavengers still present) with end over end shaking to mimic purification of tissue homogenate with antibody conjugated beads. After overnight incubation, the samples were buffer exchanged into HPLC grade water and directly infused into a 9.4T FTMS. In all cases, SOD1 remained cysteinylated, thus the thiol scavengers used here did not appear to reverse SOD1 cysteinylation.

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