Peptide degradation: deamidation, oxidation and cleavage
Peptide degradation follows a short list of chemical routes: deamidation of asparagine and glutamine, oxidation of methionine, cysteine and tryptophan, cleavage of the backbone (in acid, fastest next to aspartic acid) and cyclization at the N-terminus. Each route leaves a mass or retention-time signature that analysis can find.
Each route is a covalent change at a particular residue, so it shows as a defined mass shift, a new HPLC peak or both. Sequence decides which routes are open; pH, temperature, moisture, metals and light set the speed. What slows them in a dry solid is covered in Storage and transit of lyophilized peptides.
Deamidation of asparagine and glutamine
Deamidation turns a side-chain amide into a carboxylic acid. Asparagine reacts through a cyclic succinimide, formed when the next residue's backbone nitrogen attacks the side-chain carbonyl and ammonia leaves. Water opens the ring to aspartic acid or isoaspartic acid, in which the chain continues through the side-chain carboxyl: a net +0.98 Da from asparagine.
The neighbouring residue, pH and temperature set the rate. In Geiger and Clarke's model hexapeptide Val-Tyr-Pro-Asn-Gly-Ala, deamidation had a half-life of 1.4 days at 37 °C and pH 7.4; replacing Gly with Leu or Pro slowed it 33- to 50-fold. Of the neighbours compared, glycine gave the fastest rate. Glutamine cannot form the five-membered succinimide and deamidates far more slowly.
Oxidation of methionine, cysteine and tryptophan
Peptide oxidation targets a few side chains. Methionine becomes the sulfoxide and, under stronger oxidants, the sulfone. Cysteine forms disulfides or, with more oxidation, sulfinic and sulfonic acids. Tryptophan gives hydroxytryptophans, N-formylkynurenine and kynurenine.
Contaminating peroxides, trace transition metals and light drive these reactions; photo-oxidation is covered in Light and peptides. PubChem's record for MOTS-c (CID 146675088) gives Met-Arg-Trp-Gln-Glu-Met-Gly-Tyr-Ile-Phe-Tyr-Pro-Arg-Lys-Leu-Arg: two methionines and one tryptophan, a statement about the sequence, not about any lot.
Cleavage and cyclization
In acid, backbone cleavage is fastest at bonds next to aspartic acid, and the Asp-Pro bond is especially labile. In Oliyai and Borchardt's model hexapeptide Val-Tyr-Pro-Asp-Gly-Ala, acid split the Asp-Gly bond, while near neutral pH the peptide isomerized to isoaspartate through a cyclic imide, aspartimide. Its ring reopens to Asp or isoAsp, and isoAsp has the parent's mass.
In diketopiperazine formation, the unprotonated N-terminal amine attacks the carbonyl of residue two, releasing the first two residues as a cyclic dipeptide and leaving a peptide two residues shorter; proline at position two favours it, and the residue before the proline changes the rate. In pyroglutamate formation, an N-terminal glutamine loses ammonia and an N-terminal glutamic acid loses water to close a five-membered ring, which blocks Edman sequencing.
Aggregation, a physical route
Aggregation breaks no covalent bond. Molecules associate through hydrophobic contact, hydrogen bonding and charge, depending on concentration, pH and temperature. It leaves each molecule's mass unchanged, and the acidic organic solvents of reverse-phase HPLC can dissociate aggregates, so size-exclusion chromatography or light scattering is used instead.
Peptide degradation signatures at a glance
| Route | Residues | Mass change (monoisotopic) | How it shows up |
|---|---|---|---|
| Deamidation | Asn (fastest before Gly), Gln | +0.98 Da | High-resolution MS: 0.02 Da from the parent's 13C peak |
| Aspartimide, isoAsp | Asp, Asn | Imide −18.01 Da (Asp), −17.03 Da (Asn); isoAsp same as Asp | Imide has its own mass; isoAsp needs HPLC or MS/MS |
| Methionine oxidation | Met | +15.99 Da (sulfoxide), +31.99 Da (sulfone) | HPLC: usually earlier |
| Cysteine oxidation | Cys | −2.02 Da (disulfide); +31.99, +47.98 Da (sulfinic, sulfonic) | Dimer at 2M − 2.02 Da |
| Tryptophan oxidation | Trp | +15.99 Da, +31.99 Da, +3.99 Da (kynurenine) | MS: +16, +32, +4 |
| Cleavage | Asp-Pro, Asp-Gly | Two fragments summing to parent +18.01 Da | MS: two smaller species |
| Diketopiperazine | Residues 1 and 2 | Parent minus two residues | MS: two-residue deletion |
| Pyroglutamate | N-terminal Gln, Glu | −17.03 Da (Gln), −18.01 Da (Glu) | MS: −17 or −18; blocked to Edman |
| Aggregation | Any | None | Size-exclusion, light scattering |
What this does not establish
- Rates in the literature belong to particular sequences and conditions, so none predicts peptide stability for another peptide or for a dry solid.
- No Solara lot is shown here to have changed or not changed; that takes a measurement on the lot.
- Nothing here describes any biological property.
Questions
What is deamidation?
What is methionine oxidation in peptides?
What is a diketopiperazine?
References
- Geiger T, Clarke S. Deamidation, isomerization, and racemization at asparaginyl and aspartyl residues in peptides. Succinimide-linked reactions that contribute to protein degradation. Journal of Biological Chemistry, 1987.
- Li S et al. Chemical instability of protein pharmaceuticals: Mechanisms of oxidation and strategies for stabilization. Biotechnology and Bioengineering, 1995.
- Oliyai C, Borchardt RT. Chemical pathways of peptide degradation. IV. Pathways, kinetics, and mechanism of degradation of an aspartyl residue in a model hexapeptide. Pharmaceutical Research, 1993.
- Goolcharran C, Borchardt RT. Kinetics of diketopiperazine formation using model peptides. Journal of Pharmaceutical Sciences, 1998.
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