Cys over-oxidation (sulfenic / sulfinic / sulfonic)
Over-oxidation past disulfide: Cys → sulfenic (+16), sulfinic (+32), or sulfonic (+48) acid. Only +16 is reversible; +32 mostly stable; +48 is permanent.
- +31.9898 — +32 Da, Cys → sulfinic acid (CysO₂H)
- +47.9847 — +48 Da, Cys → sulfonic acid (CysO₃H, irreversible)
Why it happens (mechanism)
Cys-SH oxidizes to S-OH (sulfenic, +16), S-O-OH (sulfinic, +32), S-O-O-OH (sulfonic, +48) under increasingly strong oxidants. Cys sulfenic is unstable and either disproportionates back to Cys + sulfinic, or reacts with another thiol to form a disulfide. Sulfinic/sulfonic acid is mostly a dead-end.
When it strikes (triggers)
H₂O₂ (>0.01% during disulfide formation overshoots). Performic acid (used to deliberately destroy disulfides). Air + transition-metal contamination (Cu, Fe). Hot DMSO + acid.
How to spot it (MS signature)
+16, +32, +48 Da on a Cys residue. Sulfonic acid (+48) is a permanent stop; very polar — major shift in HPLC retention.
How to prevent it
- Use DMSO at 5-20% instead of H₂O₂ for disulfide formation — much gentler, kinetics controllable.
- Add EDTA (1 mM) to disulfide-formation buffers to chelate metals.
- If using H₂O₂, ≤2 eq, room temp, monitor by HPLC; quench with thiol after 1 h.
If it already happened (salvage)
- +16 (sulfenic) reduces back with DTT/TCEP/glutathione.
- +32 (sulfinic) — only specialized enzymes (sulfiredoxin) reduce in vivo; chemically considered permanent.
- +48 (sulfonic) — irreversible.
Source
Yi Yang, Side Reactions in Peptide Synthesis (Elsevier, 2016), Chapter 9, §9.1.