Short answer

Peptide sequences are written from the N-terminus to the C-terminus, using one-letter or three-letter codes. Prefixes such as Ac- describe an N-terminal modification, suffixes such as -NH2 describe a C-terminal amide, lower-case or D- marks a D-amino acid, and brackets or cyclo[...] describe unusual residues and rings.

Each of these features changes the mass, so a notation has to be read in full before an expected molecular weight is calculated.

Key facts

  • The sequence is always written from the free amino end (N-terminus) to the carboxyl end (C-terminus).
  • A C-terminal amide (-NH2) is 0.984 Da lighter than a free acid, and an N-terminal acetyl (Ac-) adds 42.011 Da.
  • D-amino acids have the same mass as L-amino acids, so they are invisible in a standard mass spectrum.
  • Lactam and disulfide rings remove 18.011 Da and 2.016 Da respectively.
  • Non-standard residues such as Aib, Nle, 2Nal and Dmt need their own residue masses.

One-letter and three-letter codes

The 20 standard amino acids have a three-letter code (Gly, Ala, Lys) and a one-letter code (G, A, K). Short peptides are often written with hyphens between three-letter codes, as in Gly-His-Lys, while longer peptides are written as strings of one-letter codes, as in YADAIFTNSYRKVLGQLSARKLLQDIMSR. Both notations describe the same chain; the string always starts at the N-terminus.

Terminal groups: free, acetylated, amidated

Unless stated otherwise, a peptide has a free amino group at the N-terminus and a free carboxylic acid at the C-terminus. Ac- in front of the sequence means that the N-terminus is acetylated, as in thymosin alpha 1 and Melanotan I. -NH2 after the sequence means that the C-terminus is an amide, as in ipamorelin, kisspeptin-10 and sermorelin.

An amide is 0.984 Da lighter than the acid, so two peptides with the same letters can differ by that amount.

D-amino acids and mixed chirality

Most natural peptides are built from L-amino acids. Synthetic peptides often include D-amino acids to resist enzymes: ipamorelin contains D-2-naphthylalanine and D-phenylalanine, GHRP-6 contains D-tryptophan and D-phenylalanine, and SS-31 contains D-arginine. In notation, D- precedes the residue or a lower-case letter is used.

Because a D-amino acid has the same mass as its L-form, mass spectrometry cannot see the difference; chiral chromatography or derivatisation methods can.

Non-standard residues

Several common research peptides use residues outside the standard 20. Aib is α-aminoisobutyric acid and appears in semaglutide, tirzepatide and ipamorelin. Nle is norleucine, a non-oxidisable replacement for methionine used in the melanotans. 2Nal is 2-naphthylalanine, Dmt is 2',6'-dimethyltyrosine, and pGlu is pyroglutamate.

Each has its own residue mass, and a calculation that treats them as their closest standard letter will not match the measured mass.

Side-chain modifications

Lipidated peptides carry a fatty acid on a lysine side chain through a spacer. This is written in parentheses after the lysine, for example Lys(γGlu-C18 diacid). The attached group adds several hundred daltons, so the mass of semaglutide or liraglutide is much higher than that of the unmodified backbone.

For these compounds, the formula published in a chemical database is the reliable source for the expected mass, and the sequence letters alone are not.

A worked reading

Melanotan II is written Ac-Nle-cyclo[Asp-His-D-Phe-Arg-Trp-Lys]-NH2. Read from the left: an acetylated N-terminus (+42.011), a norleucine, then a ring through the side chains of Asp and Lys (-18.011) containing His, D-Phe, Arg and Trp, and finally a C-terminal amide. The calculated formula is C50H69N15O9 and the monoisotopic mass is 1023.540 Da, which agrees with the public record.

Frequently asked questions

Why does a lower-case letter sometimes appear in a sequence?

Many authors use lower-case letters for D-amino acids, so f means D-phenylalanine. Always check the key that comes with the sequence.

What does the number in a name such as GRF(1-29) mean?

It gives the residues of the parent protein that are included. GRF(1-29) is the first 29 amino acids of growth hormone-releasing factor.

How do I know if a peptide is cyclic from its name?

Look for the words cyclo, a bracket that encloses part of the sequence, or a statement of a disulfide or lactam bond. Without them the peptide is assumed to be linear.

Primary references

  1. IUPAC-IUB: Nomenclature and symbolism for amino acids and peptides
  2. PubChem: Compound records for peptide structures

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