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A peptide does not «go bad» at random: it degrades through specific chemical reactions that occur at specific residues. Knowing them makes it possible to predict which peptides are fragile and why they are stored the way they are.
Deamidation
Asparagine (Asn) and, to a lesser extent, glutamine (Gln) lose their amide group and become aspartate or glutamate, with a mass increase of ~1 Da and a change in charge. It is especially fast in the Asn-Gly sequence, at neutral or alkaline pH and at elevated temperature. It proceeds through a cyclic intermediate (succinimide) that also produces isoaspartate.
Oxidation
Methionine oxidizes to sulfoxide (+16 Da); tryptophan, histidine and free cysteine are also sensitive. It is favored by dissolved oxygen, light and traces of metals. That is why some analogs replace methionine with norleucine, and why protection from light is not a minor detail.
Aspartate isomerization
Aspartate (Asp), especially in the Asp-Gly sequence, can isomerize to isoaspartate through the same succinimide intermediate. The mass does not change, but the structure does, and with it the activity.
Hydrolysis of the peptide bond
The amide bond is stable, but it breaks more easily in acidic medium and, typically, at bonds adjacent to aspartate (Asp-Pro is particularly labile).
Diketopiperazine formation
The first two residues at the N-terminus can cyclize and split off from the rest of the chain, especially if the second is proline or glycine.
| Reaction | Sensitive residues | Favored by | Mass change |
|---|---|---|---|
| Deamidation | Asn (Asn-Gly), Gln | Neutral/alkaline pH, heat | +1 Da |
| Oxidation | Met, Trp, Cys, His | Oxygen, light, metals | +16 Da |
| Isomerization | Asp (Asp-Gly) | Moderately acidic pH, heat | None |
| Hydrolysis | Bonds next to Asp | Acidic pH, heat | Fragmentation |
Almost all these reactions require water and are accelerated by temperature. That is why a dry, cold peptide lasts years and a peptide in solution at room temperature lasts days.
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