Oxidation-prone residues in peptide solutions: Met, Cys and Trp

What happens to each residue

• Methionine → methionine sulfoxide (+16 Da), and further to the sulfone (+32 Da).
• Cysteine → disulfide (inter- or intramolecular; −2 Da per bond) or sulfenic/sulfinic/sulfonic acids.
• Tryptophan → products including N-formylkynurenine (+32 Da) and kynurenine (+4 Da), promoted by light.
• Histidine and tyrosine can also oxidise, mainly under metal- or light-catalysed conditions.

Source: Stability of protein pharmaceuticals: an update, Pharm Res 2010 (PubMed 20143256); Photosensitised oxidation of tryptophan to N-formylkynurenine, Chem Res Toxicol 2024 (PubMed 39105764)

Why it matters in an assay

A single methionine oxidation can change how a peptide binds its target; a study of parathyroid hormone analogues found ligand-dependent effects of oxidising one methionine. In practice, an oxidised fraction in a stock solution is an uncontrolled variable.

Source: Ligand-dependent effects of methionine-8 oxidation in PTH analogues, Endocrinology 2021 (PubMed 33242090)

Handling steps that limit oxidation

• Prepare solutions in degassed or freshly prepared buffer; minimise headspace.
• Protect tryptophan-containing peptides from light.
• Avoid trace metals; use high-purity water and clean glassware.
• Aliquot stocks so each portion is opened once; store frozen.
• Record the lot number and preparation date for every stock.

Detecting oxidation

LC-MS shows oxidised species as peaks at +16 or +32 Da that usually elute slightly earlier than the parent on reversed-phase HPLC. Comparing a fresh solution with an aged one is a simple stability check for your own conditions. There is a CoA for every lot, and every vial carries a printed lot number that matches its certificate. A CoA is emailed within 2 hours of a request.

Frequently asked questions