Common Peptide Degradation Products and the Mass Shifts That Reveal Them

Why mass shifts matter

A purity percentage tells you how much of the sample is the main peak. It does not tell you what the minor peaks are. Mass spectrometry connects each peak to a mass, and a list of well-known mass shifts then gives a first explanation.

A minor species that is 15.995 Da heavier than the main peak is almost certainly an oxidised form; one that is 0.984 Da heavier is probably deamidated; one that is 2.016 Da heavier may be a reduced disulfide. The explanation is a hypothesis to test, but it points the investigation in the right direction.

Oxidation of methionine, tryptophan and cysteine

Methionine is the most easily oxidised residue and converts to methionine sulfoxide (+15.995 Da); stronger oxidation gives the sulfone (+31.990 Da). Peptides such as Semax, MOTS-c, sermorelin, tesamorelin and thymosin beta-4 contain methionine. Tryptophan also oxidises, giving +15.995, +31.990 and related products, and cysteine can form sulfenic, sulfinic or sulfonic acids.

Oxidation is accelerated by light, metal ions and peroxides in solvents and excipients.

Deamidation and aspartate isomerisation

Asparagine and glutamine side chains lose ammonia and gain water to become aspartic and glutamic acid (+0.984 Da). The reaction passes through a cyclic imide intermediate that can open to the normal or to the iso form, so the same mass can correspond to two different species that elute at different retention times. Asparagine followed by glycine or serine deamidates fastest.

The imide itself is 17.027 Da lighter than the intact peptide.

Disulfide bonds: reduced, scrambled and dimerised

Each intramolecular disulfide bond lowers the mass by 2.016 Da compared with the two free thiols. An open (reduced) form is therefore 2.016 Da heavier than the intact peptide, as is the case for oxytocin, AOD9604 and cagrilintide. Peptides with several disulfides, such as IGF-1 LR3, can also form wrongly paired isomers that have the same mass as the correct molecule and differ only in retention time.

Free thiols can combine to form dimers with a mass of 2M - 2.016.

Pyroglutamate and loss of water

An N-terminal glutamine cyclises to pyroglutamate with a loss of ammonia (-17.027 Da), and an N-terminal glutamic acid does the same with a loss of water (-18.011 Da). Gonadorelin and ARA290 are written with pyroglutamate on purpose.

Elsewhere in a sequence, a loss of 18.011 Da can indicate dehydration of aspartic acid to an anhydride or imide, or incomplete ring closure in a cyclic peptide that is expected to be lactamised.

Adducts and other artefacts of the measurement

Sodium and potassium ions from glassware or buffers form [M+Na]+ and [M+K]+ ions, which sit 21.982 and 37.956 Da above [M+H]+. Formic acid in the mobile phase can add a formyl group (+27.995). Trifluoroacetate can appear as an adduct of the protonated peptide. These species come from the way the sample was measured and do not indicate degradation.

Truncation, deletion and insertion

Solid-phase synthesis can produce peptides in which one amino acid is missing (deletion) or an extra amino acid is added. The mass shift equals the residue mass of the amino acid involved: 57.021 for glycine, 71.037 for alanine, 99.068 for valine and 128.095 for lysine.

These impurities are often close to the main peak in a chromatogram, so a stability-indicating, high-resolution HPLC method is needed to separate them.