A peptide that gave clean data in March and ambiguous data in September has usually changed, not misbehaved. Degradation in research peptides is chemistry, it is predictable from the sequence, and most of it is avoidable. This page covers which residues are vulnerable, what each reaction does to the mass, and what to change — in the sequence, the salt form or the storage — when a peptide will not stay intact.
| Reaction | Residues | Mass change | What triggers it |
|---|---|---|---|
| Oxidation to the sulfoxide | Met | +16 Da | Air, light, trace metals, peroxides in old solvent |
| Disulfide formation | two Cys | −2 Da | Dissolved oxygen, neutral to basic pH |
| Oxidation | Trp, and Cys beyond the disulfide | +16 Da and higher | Light, air, metals |
| Deamidation | Asn, and more slowly Gln | +1 Da | Neutral to basic pH; fastest when followed by Gly |
| Acid-catalysed cleavage | Asp-Pro | chain breaks | Low pH, warmth, long storage in solution |
| Pyroglutamate formation | N-terminal Gln or Glu | −17 or −18 Da | Spontaneous, faster when warm |
| Aggregation | hydrophobic and β-sheet-prone sequences | none | Concentration, freeze-thaw, time |
The small mass shifts are worth memorising, because they turn an unexplained shoulder on a chromatogram into a diagnosis. A +16 peak on a methionine-containing peptide is oxidation; a +1 on an Asn-Gly sequence is deamidation. Our modification mass table lists the shifts for deliberate modifications alongside these.
Asparagine deamidation is the most commonly overlooked degradation route in research peptides, for three reasons. The mass change is only +1 Da, which is easy to miss. The rate depends strongly on the residue that follows: Asn-Gly is markedly faster than Asn followed by a bulky residue. And the product is not a single species, because the reaction proceeds through a cyclic intermediate that can open either way, giving both the expected aspartate and an isoaspartate with an altered backbone. An isoaspartate peptide can lose antibody recognition or activity while looking almost unchanged by routine analysis.
If your sequence contains Asn-Gly and the peptide will be in solution for any length of time, that is worth knowing before the experiment rather than after.
Keep the lyophilized powder cold and dry, and warm the vial to room temperature before opening so moisture does not condense into it. Make a concentrated stock, split it into single-use aliquots and freeze those, rather than thawing one tube repeatedly. See storage and handling and aliquoting.
Mildly acidic conditions slow deamidation; strongly acidic conditions accelerate Asp-Pro cleavage, so the two constraints pull in opposite directions and the right pH depends on which residues you have. Degassing helps with cysteine and methionine. Keep oxidation-prone peptides out of the light.
Where the assay allows it, a conservative substitution removes the problem permanently: Asn to Gln or Ser, Met to norleucine, or breaking an Asn-Gly by changing the glycine. Any substitution has to be checked against activity, so make the parent alongside.
Peptides purified by reverse-phase HPLC carry residual TFA, which lowers the pH of a concentrated stock and can affect both stability and cell assays. Exchange is available — see TFA removal and salt conversion.
The commonest avoidable loss
Repeated freeze-thaw of one stock tube. Each cycle concentrates solutes at the ice boundary and gives aggregation-prone sequences another chance to nucleate. Aliquoting once, on the day the peptide arrives, prevents more degradation than any buffer choice.
Usually the peptide has changed. Oxidation, deamidation and aggregation all proceed slowly in storage. Re-run the HPLC against the trace supplied with the lot and check the mass for shifts of +16, +1 or minus 2 Da.
Methionine and cysteine oxidise, tryptophan oxidises under light, asparagine deamidates (fastest when followed by glycine), an Asp-Pro bond cleaves under acid, and an N-terminal Gln or Glu can cyclise to pyroglutamate. Hydrophobic and beta-sheet-prone sequences aggregate.
Oxidation of methionine to the sulfoxide adds 16 Da. Disulfide formation between two cysteines removes 2 Da. Deamidation adds 1 Da. Aggregation produces no mass change at all, which is why it is often missed.
Aliquot the stock into single-use tubes on the day the peptide arrives. Repeated freeze-thaw of one tube causes more avoidable degradation than any buffer choice.
Within three months of shipment, yes. A retained sample of each lot is kept for that period, so the original material can be compared against yours.
Research use only. LifeTein peptides and proteins are supplied for laboratory research use. They are not for human or veterinary use, and not for diagnostic or therapeutic use. See our research use policy.