Common Peptide Impurities and Degradation Pathways

Impurities from the synthesis

• Deletion sequences: chains missing one residue because a coupling step did not go to completion.
• Truncated sequences: chains that stopped growing early.
• Incompletely deprotected peptides: chains still carrying a protecting group.
• Diastereomers: peptides in which one amino acid has flipped to its mirror-image form.
• Residual solvents and reagents, and counter-ions such as trifluoroacetate.

Changes over time

• Hydrolysis: water breaks peptide bonds, which is a main reason peptides are stored dry. Some sequences are more prone than others.
• Deamidation: asparagine and glutamine lose an amide group, which changes the mass by about one dalton.
• Oxidation: methionine, cysteine and tryptophan are the most susceptible. An oxidised methionine adds 16 daltons.
• Aggregation: peptide molecules can clump together, especially in solution.
• Cyclisation: an N-terminal glutamine can convert to pyroglutamate.

How these show up on a report

Many appear as small extra peaks beside the main peak on an HPLC chromatogram. Mass spectrometry can reveal species whose mass differs from the target by a characteristic amount, such as +16 for an oxidised methionine. Neither method finds everything, which is why reports state what was measured. See How Peptide Purity Is Tested.

Slowing degradation

Store the sealed vial dry, cold and dark, let it reach room temperature before opening, and avoid repeated warm-and-cold cycles. See How to Store Lyophilized Research Peptides.