How to Read Peptide Sequences: Codes, Termini, Modifications and Cyclic Peptides

A peptide sequence is a list of amino-acid symbols written from the N-terminus to the C-terminus, with prefixes, suffixes and brackets for anything that is not a plain linear chain of L-amino acids. Read correctly, a line such as Ac-Nle-cyclo[Asp-His-D-Phe-Arg-Trp-Lys]-NH2 gives the structure, the expected mass and several clues to the chemistry. The symbols and conventions used here follow the IUPAC-IUB recommendations for amino acids and peptides ^[1], with the informal variants that appear on supplier listings noted where they differ.

The two alphabets

Each of the 20 standard amino acids has a three-letter code and a one-letter code. Three-letter codes are joined by hyphens (Gly-His-Lys); one-letter codes are written as a continuous string (GHK). Both describe the same peptide sequence.

The letters that do not match the name are the usual trip-ups: F is Phe, Y is Tyr, W is Trp, K is Lys, R is Arg, D is Asp, E is Glu, N is Asn and Q is Gln. Residue masses are the free amino acid minus one water, because each peptide bond releases water; a peptide's mass is the sum of its residues plus one water for the free termini.

Direction, numbering and the termini

Sequences are always written N-terminus first. Position 1 is the residue with the free amino group, and numbers count towards the C-terminus.

• H- marks a free N-terminal amine and -OH a free C-terminal acid. Both are often left implicit.
• Ac- is an N-terminal acetyl group, adding C2H2O (+42.011 Da).
• -NH2 is a C-terminal amide, replacing OH with NH2 (−0.984 Da). Many signalling peptides are amidated, and the difference is visible by mass spectrometry: a confiscated GHRH analogue was found by high-resolution MS to be only partly amidated, which the analysts read as a sign of poor quality ^[2].

Fragment notation gives the numbered range of a parent sequence: GRH(1–29)-NH2 is the first 29 residues of growth hormone-releasing hormone with an amidated C-terminus, and GRH(1–40)-OH a 40-residue form with a free acid ^[3]. Numbering always follows the parent molecule. GLP-1 analogues are numbered from 7, because the circulating active hormone is GLP-1(7–36)amide ^[4]; semaglutide's "Aib8" is therefore the second residue of the chain, which is exactly where DPP-IV cuts ^{[4, 5]}.

Substitutions are written in square brackets before the parent name. [Pro1, Val14]-hGHRH, for example, is human GHRH with proline at position 1 and valine at position 14; in that confiscated sample the "Pro1" was in fact an added N-terminal proline, an example of why the bracket shorthand needs checking against the full sequence ^[2].

Stereochemistry: D-amino acids

Every standard residue except glycine is chiral, and L is assumed unless stated. A D-residue is written with a D- prefix: the growth hormone-releasing hexapeptide GHRP is His-D-Trp-Ala-Trp-D-Phe-Lys-NH2 ^[6]. Informal one-letter strings sometimes use lower-case letters for D-residues, and some database synonyms drop stereochemistry altogether. PubChem, for instance, lists ipamorelin both with and without its D- prefixes.

D- and L-forms have identical masses, so mass spectrometry cannot tell them apart. Racemisation during synthesis produces exactly this kind of diastereomeric impurity ^[7], which is why a stereochemical impurity shows up only as a separate HPLC peak. D-residues are used deliberately to resist proteolysis; peptide half-life research covers that chemistry.

Non-standard residues

Many research peptides contain residues outside the standard 20. They have no one-letter code and are written with their abbreviations inside a three-letter sequence.

Side-chain and terminal modifications

Modifications attached to a residue are written in brackets after it, or described in words when the group is large.

• Side-chain acylation. Semaglutide is derivatised at Lys26 ^[5] with an 18-carbon fatty diacid linked through γ-glutamate and two short ethylene-glycol spacers, often abbreviated Lys26(…) in sequence diagrams.
• Reactive groups. CJC-1295 with DAC carries an added C-terminal lysine bearing a 3-maleimidopropionamide group on its ε-amine ^[9].
• N-terminal acyl groups other than acetyl. Tesamorelin is human GHRH(1–44)NH2 with a trans-3-hexenoyl group on Tyr1 ^[10].
• Pyroglutamate. An N-terminal Gln or Glu can cyclise to pyroglutamate (pGlu), one of the degradation products seen in peptide medicines ^[7].
• Metal complexes. GHK-Cu is the tripeptide GHK complexed with copper(II) ^[11]; the metal is noted after the sequence rather than within it.

Cyclic peptides and disulfides

Cyclisation is written by enclosing the ring in brackets, with cyclo or c, and the type of link stated or implied.

Side-chain lactam. Melanotan II is Ac-Nle-cyclo[Asp-His-D-Phe-Arg-Trp-Lys]-NH2: an amide bond joins the side-chain carboxyl of Asp2 to the side-chain amine of Lys7, closing a six-residue ring and releasing one water (−18.011 Da). PT-141 (bremelanotide), a synthetic α-MSH analogue ^[12], has the same ring with a free C-terminal acid instead of the amide. That one change is the whole structural difference between them: C50H69N15O9 (1024.2 g/mol) for Melanotan II against C50H68N14O10 (1025.2 g/mol) for PT-141. A forensic laboratory characterising seized samples distinguished the two by accurate mass and fragmentation ^[13].

Disulfide bridge. Two cysteine thiols oxidise to a disulfide, removing two hydrogens (−2.016 Da). None of the catalogue peptides contain cysteine, so the standard textbook example is oxytocin, CYIQNCPLG-NH2 with a Cys1–Cys6 disulfide, written as cyclo(1–6) or with a line joining the two cysteines. Its formula, C43H66N12O12S2 (1007.2 g/mol), is two hydrogens lighter than the reduced form.

Worked examples from the catalogue

Formulas and masses were calculated from the sequences and cross-checked against PubChem records where they exist. Ipamorelin is a useful exercise. It came from a series of compounds lacking the central Ala-Trp dipeptide of GHRP-1 ^[8], and adding the residue formulas of Aib, His, D-2-Nal, D-Phe and Lys, plus water, minus the O-to-NH change of the amide, gives C38H49N9O5 exactly.

Mass shifts worth memorising

These shifts, together with the residue table above, are what a mass spectrometrist uses to assign unexpected peaks; what HPLC testing measures covers the complementary separation side.

For the bench

• Paste standard sequences into the peptide molecular weight calculator, which handles N-acetyl, C-amide and disulfides; enter L for Nle, and add Aib, Nal or lactam corrections by hand.
• Use the average molecular weight for weighing and molarity, and the monoisotopic mass for interpreting spectra; the molarity calculator needs the average figure.
• Compare the sequence on the vial label, the certificate and the literature you are following, including termini and stereochemistry.
• Remember that sequence notation describes the peptide, not the powder: counter-ions and water are additional, as net peptide content explains, and how peptides are made shows where those come from.
• For what these reagents are and how they differ from medicines, see what are research peptides.