Commonly Used Alkylating Agents Limit Persulfide Detection by Converting Protein Persulfides into Thioethers

Area of Science:

• Biochemistry
• Molecular Biology
• Post-Translational Modifications

Background:

• Protein persulfides (R-S-SH) are increasingly recognized as critical post-translational modifications.
• Accurate detection and quantitation of protein persulfides are essential for understanding their biological roles.
• Current methods rely on trapping persulfides with alkylating agents prior to analysis, typically by mass spectrometry.

Purpose of the Study:

• To evaluate the efficacy of various alkylating agents in preserving the persulfide sulfur-sulfur bond.
• To identify alkylating agents suitable for the stable detection of protein persulfides.
• To elucidate the chemical mechanisms underlying the stability or instability of persulfide adducts.

Main Methods:

• Incubation of persulfide-containing peptides with different alkylating agents (N-ethylmaleimide, iodoacetamide, monobromobimane, N-t-butyl-iodoacetamide).
• Analysis of the resulting adducts using mass spectrometry to determine their stability.
• Investigation of reaction pathways involving tautomerization and nucleophilic attack.

Main Results:

• N-ethylmaleimide and iodoacetamide yield unstable persulfide adducts (R-S-S-Alk) that readily convert to thioethers (R-S-Alk).
• Monobromobimane and N-t-butyl-iodoacetamide successfully generate stable alkylated persulfides (R-S-S-Alk).
• The stability of the persulfide adduct is dependent on the chemical properties of the alkylating agent, influencing tautomerization and sulfur donation.

Conclusions:

• The choice of alkylating agent is critical for the accurate detection and quantitation of protein persulfides.
• Commonly used agents like N-ethylmaleimide and iodoacetamide are not ideal for preserving persulfide integrity.
• Monobromobimane and N-t-butyl-iodoacetamide represent superior choices for conserving persulfides for mass spectrometry-based analyses.