Protocol: Reduction and Alkylation of Disulfide-Rich Peptides or Proteins
To reduce disulfide bonds in a cysteine-rich peptide or proteins and subsequently alkylate the free thiol groups to prevent reformation, enabling assessment of disulfide bond contribution to antimicrobial activity. This reaction converts cys into carboxyamidomethylcysteine when using iodoacetamide.
Notes
Antimicrobial peptide (lyophilized) >95% purity recommended. 1μg/μL
Dithiothreitol (DTT) or TCEP: Reducing agent
Iodoacetamide (IAA): Alkylating agent
100 mM Ammonium bicarbonate (ABC) pH 8.0
50 mM HEPES pH 8.2
Urea Optional, for solubilization (6 M)
Water (Milli-Q) Sterile
Light-protected tubes: For IAA reactions
Incubator / water bath (37°C): For reduction
Mass spectrometry or HPLC: Optional, for confirmation
· Dissolve peptide in 100 mM ABC buffer (pH 8.0) to a concentration of 0.1–1 mg/mL.
· (alternative) Dissolve peptide in 50 mM HEPES buffer (pH 8.2) to a concentration of 1 mg/mL.
Solution pH is very important for alkyation reaction. Avoid acidic conditions during IAA reaction (prevents side reactions).
Optionally, add 6 M urea (final concentration) for better solubility of hydrophobic peptides.
· Add DTT to a final concentration of 10 mM (or TCEP at 5–10 mM). Incubate the mixture at 37°C for 1 hour. Ensure tube is sealed and vortexed briefly before incubation.
· Cool the sample to room temperature. Add iodoacetamide (IAA) to a final concentration of 20 mM. Incubate in the dark at room temperature for 1 hour.
· Add additional DTT (10 mM) to quench excess IAA. Incubate in the dark for another 15 minutes.
· Desalt or remove excess reagents using dialysis, spin column, or HPLC purification.
· Confirm reduction and alkylation by mass spectrometry (mass shift of +57 Da per cysteine) or HPLC.
Once reduced and alkylated, perform MIC assay, zone of inhibition, or bacterial killing curves to compare native vs. reduced/alkylated peptide.
· If using TCEP, it does not need to be removed before alkylation unlike DTT.