Peptide Sequence Notation: How to Read a Written Peptide

Why the notation matters

Most laboratory records describe a peptide with a single line of letters and symbols. That line has to carry a lot: the amino acid sequence, any changes to the ends of the chain, the configuration of each residue and any ring or bridge. Every later calculation, from molecular weight to the masses expected in a spectrum, starts from that line. A misread hyphen or a missed lowercase letter changes the molecule and every number derived from it.

The conventions below follow the long-standing biochemical nomenclature recommendations of the IUPAC-IUB Joint Commission on Biochemical Nomenclature, plus a few common extensions used in synthesis and proteomics. Where a document uses its own convention, it should say so; when it does not, the standard reading described here is the sensible default, and anything ambiguous is a question to put to whoever wrote it.

Three-letter and one-letter codes

Each of the twenty standard amino acids has a three-letter abbreviation and a one-letter code. The three-letter form is easier to read and is usual in synthesis and in short sequences; the one-letter form is compact and is the norm in databases and software. A tetrapeptide can be written Gly-His-Lys-Ala or GHKA; both describe the same chain.

• Hyphens join residues in three-letter form. Gly-Pro-Gln means three residues linked by two peptide bonds. In one-letter form the letters are simply run together: GPQ.
• Some one-letter codes are not the initial letter. K is lysine, R arginine, D aspartic acid, E glutamic acid, N asparagine, Q glutamine, F phenylalanine, W tryptophan and Y tyrosine. Mixing up D and N, or E and Q, changes a residue by about one mass unit and swaps an acid for an amide.
• B, Z and X mean uncertainty. B stands for aspartic acid or asparagine and Z for glutamic acid or glutamine when the two cannot be told apart; X means any or unknown residue. They belong in analytical reports, not in the specification of a synthetic material.
• Non-standard residues have their own abbreviations. Synthetic sequences often contain residues outside the standard twenty, for example Nle (norleucine), Aib (2-aminoisobutyric acid), Orn (ornithine) or Nal (naphthylalanine). These have no agreed one-letter code, so they are usually written out in three-letter form or placed in brackets.

Direction: N-terminus to C-terminus

By convention a sequence is written from the amino terminus on the left to the carboxyl terminus on the right. Gly-His-Lys and Lys-His-Gly are different molecules with the same composition and the same mass; only the direction tells them apart.

A fully written free peptide is sometimes shown as H-Gly-His-Lys-OH. The H- on the left is the hydrogen of the free amino group and the -OH on the right is the hydroxyl of the free carboxyl group. When these end groups are left off, the free forms are assumed. When something else appears in their place, the ends have been modified.

Terminal groups: Ac- and -NH2

Two end modifications are common enough that they appear in many catalogue sequences. Ac- at the left means the amino terminus carries an acetyl group (acetylation). -NH2 at the right means the carboxyl terminus is an amide rather than an acid (amidation). A chain written Ac-Ser-Tyr-Ser-NH2 is capped at both ends.

Each change has a fixed effect on mass. Acetylation adds C2H2O, which is about 42.011 Da monoisotopic. Amidation replaces an OH with NH2, a change of about −0.984 Da. The amidation shift is small enough to be overlooked in a quick check, yet it is close to the shift caused by deamidation of an asparagine, which is why the written form of the C-terminus has to be read carefully before a measured mass is compared with a calculated one. Charge changes too: an acetylated amino terminus and an amidated carboxyl terminus no longer carry a charge, which shifts the calculated isoelectric point. See the peptide properties calculator.

Stereochemistry: L, D and lowercase letters

Amino acids other than glycine are chiral. The standard residues in natural proteins are the L forms, and an unmarked residue is read as L. A D-residue is marked with a prefix, as in D-Phe, and some software and papers use a lowercase one-letter code for it, so f means D-phenylalanine where F means L-phenylalanine. That makes letter case meaningful: a sequence pasted into a tool that converts everything to capitals has silently changed.

D- and L-forms have identical masses, so a mass spectrum alone cannot say which is present. Separating such isomers needs methods that respond to shape, such as chiral chromatography or ion mobility; a review of D-amino-acid-containing peptides describes how ion mobility-mass spectrometry separates them and can locate the D-residue from fragment arrival times.^[1] Conversion of an L-residue to D during synthesis is called racemization, one of the impurity types discussed in the synthesis overview.

Rings and bridges

A ring closed through the backbone is usually written cyclo(...) or cyclo[...], with the residues inside the brackets. Cyclization from head to tail removes one water, about 18.011 Da monoisotopic, compared with the open chain. Side-chain rings, such as a lactam between an aspartic acid and a lysine, are written by placing only the ring residues inside the brackets, so a sequence can be part linear and part cyclic.

A disulfide bond between two cysteines is commonly shown with a note such as (Cys2–Cys7 disulfide) or with a line drawn between the two residues. Forming each disulfide removes two hydrogens, about 2.016 Da, so the oxidized and reduced forms differ by that amount per bridge. Cyclic peptides also complicate fragment spectra, because a ring must break twice before a piece can leave; a nomenclature for labeling cyclic peptide fragments uses the two residues on either side of the first broken bond in each label.^[2]

Notation in mass spectrometry

The same sequence notation is used to name fragments in tandem mass spectrometry. In the scheme proposed by Roepstorff and Fohlman, fragments that keep the amino terminus are labeled a, b or c and those that keep the carboxyl terminus x, y or z, with a number giving how many residues the fragment contains.^[3] A b3 ion therefore holds the first three residues from the left of the written sequence and a y3 ion the last three from the right, which is one more reason the direction of the written sequence matters.

Proteomics has also produced machine-readable notations. ProForma, proposed by the Consortium for Top-Down Proteomics, writes a sequence in standard one-letter code with modifications or unexplained mass shifts placed in square brackets after the residue they belong to, so that people and software read the same thing.^[4] A line such as EM[Oxidation]EVTK is an example of that style. For worked mass calculations see mass spectrometry for peptide identity.

What a written sequence does not tell you

A sequence describes the peptide itself, not the powder in a vial. It says nothing about the counter-ion, the water content or the purity. A specification that adds a word such as acetate or trifluoroacetate is describing the salt form; see TFA and acetate counter-ions and purity versus net peptide content. The molecular weight calculated from the sequence is therefore the mass of the peptide, and is lower than the mass of the same number of moles of the salt.

A checklist for reading any sequence

1. Confirm the direction. Unless stated otherwise, the left end is the amino terminus.
2. Look at both ends for H-, Ac-, -OH or -NH2, and note any other end group.
3. Check every one-letter code against its residue, especially D and N, E and Q, and K and R.
4. Look for D- prefixes or lowercase letters and keep the case when copying the sequence into software.
5. Identify any cyclo notation or disulfide note, and count how many bridges there are.
6. Write out non-standard residues in full and confirm their masses before calculating.
7. Calculate the expected mass from the full description, not just the letters, and compare it with the stated molecular weight.

To try this on a real sequence, enter it into the peptide molecular weight estimator, or look up a term in the glossary. Questions about how a listed material is written can be sent to support.