Fig. 3: Quantification of protein persulfides (PrSS–) and polysulfides (PrS(S)nS–.
a Schematic showing expected reactions of protein thiol with Na2S2, or with DTNB followed by Na2S. b–d Reduced Trx1 (70 µg) was incubated with 0.5 mL Neutravidine-agarose bead slurry for 60 min, washed, treated with Na2S2 (0-300 µM) for 7 min (b, c) or with 20 µM DTNB for 20 min, followed by washing and exposure to Na2S (15 µM) for 7 min (d). All samples were then washed and processed by Procedure A for the generation of CAM-S-CAM-TPP (b, d) or Procedure B for S(CAM-TPP)2 (c). Indicated samples were treated with TCEP (5 mM) prior to analysis, or ZnCl2 (1 mM) was added along with the Na2S (d). Data are mean ± S.D. n = 3. e Human recombinant Cofilin 1 (11 μg) was incubated with 1 mM GSH ± MitoPerSulf (100 μM) for 7 min, and then CAM-S-CAM-TPP products were quantified by Procedure A. HEK 293 cell lysates (100 µg protein in 200 µL 50 mM HEPES buffer pH 7.4) were exposed to Na2S (40 µM) or to Na2S2 (40-600 µM) for 7 min, alkylated with either IAM-TPP (f) or IAM (g), bound to the SP3 beads slurry and washed with HEPES buffer. Samples were then processed by procedure A (f) or procedure B (g). Data are mean ± S.D. n = 3 (n denotes the number of individual technical replicates). Selected protein structure elements in (a) were prepared with the assistance of ChemDraw software (version 23.1.2). Source data are provided as a Source Data file.
