How to read a peptide sequence

A peptide sequence lists amino acids in order from the N-terminus to the C-terminus, with any changes to the ends or side chains marked around it. Read it correctly and you can confirm a compound's identity, work out its mass and check it against the lot's certificate of analysis.

Direction: N-terminus to C-terminus

Sequences are written left to right from the N-terminus, the residue with the free amino group, to the C-terminus, the residue with the free carboxyl group. BPC-157 is written GEPPPGKPADDAGLV: glycine is at the N-terminus, valine at the C-terminus, 15 residues in all.

Some notations spell the ends out, as H-Gly-Glu-…-Val-OH, where H- is the free amine and -OH the free acid. A sequence with nothing marked at either end is read as having both ends free.

Direction matters. The same letters written in reverse are a different peptide, even though the formula and mass are identical.

One-letter and three-letter codes

Each of the 20 standard amino acids has a three-letter code and a one-letter code. Three-letter sequences are joined with hyphens (Gly-Glu-Pro-Pro-Pro-Gly-Lys-Pro-Ala-Asp-Asp-Ala-Gly-Leu-Val); one-letter sequences are written as a single string. Both carry the same information.

Nine one-letter codes are not the amino acid's initial, and they are the easiest to misread:

The full list, with residue masses and side-chain charge, is in the amino acid table. X, B and Z are ambiguity codes (any residue; Asp or Asn; Glu or Gln) and have no place in the specification of a defined compound.

End caps: Ac- and -NH2

A prefix or suffix on the sequence marks a modified end:

• Ac- at the start is N-terminal acetylation: an acetyl group on the N-terminal amine, which removes the positive charge at that end and adds 42.04 Da (average mass). Thymosin Alpha-1 is acetylated: Ac-SDAAVDTSSEITTKDLKEKKEVVEEAEN.
• -NH₂ at the end is C-terminal amidation: the C-terminal acid is an amide instead, which removes the negative charge at that end and lowers the mass by 0.98 Da. Oxytocin ends in glycinamide: CYIQNCPLG-NH₂.
• H- at the start and -OH at the end mark the free amine and the free acid, the same as leaving the ends unmarked.

An amide and a free acid differ by 0.98 Da, close to the spacing of isotope peaks, so a low-resolution mass spectrum can blur the two. The specification and the lot COA should state which end you have.

Disulfide bridges

Two cysteines (C) can be joined through their side chains by a disulfide bond, which closes a loop in the chain. The bond is noted after the sequence, as in "disulfide Cys1–Cys6", or by numbering the bridged positions. Each disulfide lowers the mass by 2.02 Da, the two hydrogens lost when the bond forms.

In oxytocin, the cysteines at positions 1 and 6 are bridged, making a six-residue ring with a three-residue tail that ends in the amide. AOD-9604, YLRIVQCRSVEGSCGF, has one disulfide, between its cysteines at positions 7 and 14.

A bridge doesn't change the letters, so the sequence alone can't tell you whether the cysteines are joined. The mass can: the bridged form is 2 Da lighter than the open, reduced form.

D-amino acids and non-standard residues

Every standard amino acid except glycine comes in L and D forms, and natural peptides are built from L. A D residue is written with a D- prefix in three-letter notation (D-Ala) or, in some one-letter notations, as a lowercase letter (a). D and L forms have the same formula and mass, so the mass alone can't tell them apart; chiral analysis can.

Many research peptides also contain residues outside the standard 20, such as Aib (2-aminoisobutyric acid), Nle (norleucine), pGlu (pyroglutamic acid at the N-terminus) or Sar (sarcosine), and some carry fatty-acid or other groups on a side chain. These are written in three-letter or bracketed form inside the sequence. Their masses are not in standard residue tables, so take the formula from the supplier's specification or from PubChem; our molecular weight reference lists the formula and mass of each catalog compound.

From sequence to molecular weight

A peptide's mass is the sum of its residue masses, plus one water for the free ends, plus any modifications. For oxytocin:

• Nine residues plus one water: 1010.19 Da (average).
• C-terminal amide: −0.98 Da.
• One disulfide: −2.02 Da.
• Result: 1007.19 Da, which matches the 1007.2 g/mol in the reference table.

Average masses, from standard atomic weights, are the ones for weighing and molarity. Monoisotopic masses, from the most abundant isotope of each element, are what a mass spectrometer resolves for small peptides: 1006.44 Da for oxytocin. The peptide molecular weight calculator does this sum for any sequence of standard residues, with options for end caps and disulfides.

Checking a sequence against a COA

The mass-spec line on a certificate of analysis is the check that a lot is the sequence you expect. Electrospray instruments report ions rather than the neutral mass: [M+H]⁺ is the mass plus one proton (about 1.007 Da), and [M+2H]²⁺ is the mass plus two protons, divided by two. For oxytocin, expect about 1007.44 for [M+H]⁺ and 504.23 for [M+2H]²⁺.

A reading about 16 Da high can point to an oxidized methionine, about 1 Da high to deamidation, and 2 Da high in a bridged peptide to an open disulfide. The amino acid table lists these shifts, and how to read a peptide COA covers the rest of the certificate.

Kit pages for BPC-157, oxytocin, Thymosin Alpha-1 and other single-sequence kits list the sequence in one- and three-letter form, with its length and any disulfide.

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