Key takeaways
- Peptide sequences are always written from the N-terminus (left) to the C-terminus (right), and residue 1 is the N-terminal residue.
- Three-letter codes joined by hyphens (H-Asp-Arg-Val-OH) are explicit about termini; one-letter strings (DRV) are compact but say nothing about end groups unless annotated.
- Prefixes and suffixes such as Ac-, H-, -OH and -NH2 change the molecular formula and must match the specification exactly.
- L-configuration is assumed unless a residue is marked D-; lowercase one-letter codes for D-residues are common but not universal, so check the document's key.
- Disulfides, cyclization, pyroglutamate and non-standard residues need explicit annotation so the sequence can be checked against mass spectrometry data.
Peptide sequence notation is the shorthand that ties a material to its identity: the same string appears on the specification sheet, the certificate of analysis (COA) and the mass spectrum report. Reading it correctly means understanding amino acid codes, the direction of the chain, terminal groups and the symbols used for modifications. This guide walks through the conventions laboratory staff will meet most often and the ambiguities worth checking.
Peptide sequence notation basics: direction and numbering
By convention, a peptide is written with its free amino terminus (N-terminus) on the left and its carboxyl terminus (C-terminus) on the right. Residues are numbered from the N-terminus, so in an eight-residue peptide the N-terminal residue is position 1 and the C-terminal residue is position 8.
This direction is the reverse of the order in which most synthetic peptides are assembled. In solid-phase synthesis the C-terminal residue is anchored to the resin first and the chain grows toward the N-terminus, which is covered in our article on solid-phase peptide synthesis. The written sequence, however, always follows N-to-C order.
Why direction matters
Reversing a sequence produces a different molecule with the same composition and the same mass. A retro-sequence cannot be distinguished from the intended peptide by intact mass alone, which is one reason tandem MS fragment data or orthogonal chromatography is used when sequence order must be confirmed.
One-letter and three-letter amino acid codes
The IUPAC-IUB recommendations define both a three-letter and a one-letter symbol for each of the 20 standard amino acids. Three-letter codes are easier to read and leave room for modification symbols; one-letter codes are compact and are the standard for databases and sequence software.
| Amino acid | 3-letter | 1-letter | Amino acid | 3-letter | 1-letter |
|---|---|---|---|---|---|
| Alanine | Ala | A | Leucine | Leu | L |
| Arginine | Arg | R | Lysine | Lys | K |
| Asparagine | Asn | N | Methionine | Met | M |
| Aspartic acid | Asp | D | Phenylalanine | Phe | F |
| Cysteine | Cys | C | Proline | Pro | P |
| Glutamine | Gln | Q | Serine | Ser | S |
| Glutamic acid | Glu | E | Threonine | Thr | T |
| Glycine | Gly | G | Tryptophan | Trp | W |
| Histidine | His | H | Tyrosine | Tyr | Y |
| Isoleucine | Ile | I | Valine | Val | V |
A few additional symbols appear in older literature and in protein sequencing data: B (Asx, Asn or Asp), Z (Glx, Gln or Glu) and X (Xaa, unknown or unspecified). On a specification for a synthetic peptide, these ambiguity codes should not appear, because the sequence is defined by design.
The same peptide in both formats
Angiotensin II, a common analytical standard, illustrates the two styles:
- Three-letter: H-Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-OH
- One-letter: DRVYIHPF
In the three-letter form each hyphen between residues represents a peptide (amide) bond. The leading H- and trailing -OH state that the N-terminal amine and the C-terminal carboxylic acid are free.
N-terminal and C-terminal conventions
Terminal groups are a frequent source of transcription errors because they are easy to drop when a sequence is copied into a one-letter string. Each one changes the molecular formula, so it must be carried through to the calculated mass. Our guide to peptide molecular weight calculation shows how each group is added.
| Notation | Meaning | Monoisotopic mass change vs. free terminus |
|---|---|---|
| H- | Free N-terminal amine | 0 (reference) |
| Ac- | N-terminal acetyl group (acetylated amine) | +42.0106 Da (C2H2O) |
| pGlu- (also Pyr-, <Glu-) | N-terminal pyroglutamate (cyclized Gln or Glu) | −17.0265 Da from Gln; −18.0106 Da from Glu |
| -OH | Free C-terminal carboxylic acid | 0 (reference) |
| -NH2 | C-terminal primary amide | −0.9840 Da (O replaced by NH) |
Pyroglutamate deserves special attention. Because the N-terminal amine is consumed in forming the lactam ring, a sequence that begins with pGlu has no free H- terminus, and it will not react in Edman degradation or with amine-reactive derivatization reagents. In one-letter strings it is often written as pE or with a bracketed annotation.
Stereochemistry and non-standard residues
Unless stated otherwise, every chiral residue in a written sequence is assumed to be the L-enantiomer. D-residues are marked explicitly, for example H-Ala-D-Phe-Gly-Leu-OH. In one-letter strings many groups use lowercase letters for D-residues (AfGL), but this convention is not universal; a reliable document defines it in a footnote or key.
Non-proteinogenic residues use established three-letter abbreviations, including:
- Nle (norleucine), Orn (ornithine), Aib (α-aminoisobutyric acid) and Sar (sarcosine, N-methylglycine)
- Hyp (4-hydroxyproline) and Abu (2-aminobutyric acid)
- N-methyl residues written as MeAla, N-Me-Ala or (NMe)Ala, depending on the house style
These residues have no single-letter code in the IUPAC scheme, so one-letter strings typically show them in brackets or parentheses. When a specification uses a custom bracket notation, confirm that the molecular formula on the same document is consistent with it.
Disulfides, cyclization and side-chain modifications
Disulfide bonds
A disulfide is usually written as a note after the sequence, such as "Disulfide bridge: Cys2–Cys7" or "(2–7 S–S)". Forming each disulfide removes two hydrogen atoms (−2.0157 Da monoisotopic), so the oxidized and reduced forms differ by about 2 Da per bond, which is resolvable by most modern mass spectrometers.
Cyclic peptides
Head-to-tail cyclic peptides are written with the prefix cyclo and the sequence in parentheses, for example cyclo(-Gly-Pro-Phe-Ala-Leu-). Cyclization removes one molecule of water relative to the linear form, and the written starting residue is arbitrary, so the document should make the ring order unambiguous.
Side-chain modifications and labels
Side-chain changes are written in parentheses after the residue, for example Ser(PO3H2) or pSer for phosphoserine, Lys(Biotin) for a biotinylated lysine, and Cys(Acm) for an acetamidomethyl-protected cysteine. Labels attached to the N-terminus, such as fluorophores, are written as prefixes in the same position as Ac-.
How sequences appear on specification sheets and COAs
A well-prepared specification or certificate of analysis typically presents the sequence alongside several values that must agree with it:
- The sequence in three-letter form with explicit termini, and often the one-letter string as well.
- The molecular formula of the free peptide (without counter-ions).
- The average molecular weight and, where mass spectrometry is reported, the theoretical monoisotopic mass.
- Any disulfide pattern, cyclization or modification note.
- The salt form, such as trifluoroacetate or acetate, stated separately from the sequence.
A quick consistency check is to recalculate the monoisotopic mass from the written sequence and compare it with the theoretical value on the document and the observed value in the mass spectrometry identity report. A discrepancy of about 1 Da often points to an amide/acid mix-up; 42 Da suggests a missing acetyl group; 17 or 18 Da can indicate an unannotated pyroglutamate.
Common ambiguities to check
- Leucine and isoleucine have identical masses, so a sequence error between them will not be caught by intact mass alone.
- Spaces, line breaks or lowercase letters introduced by copying can change meaning in one-letter strings.
- "NH2" at the left of a sequence (an older style for the free N-terminus) is not the same as "-NH2" at the right (a C-terminal amide).
Frequently asked questions
Does a one-letter sequence imply a free acid C-terminus?
Not reliably. A bare one-letter string carries no information about end groups, so the terminal groups should be stated separately or the three-letter form should be used.
How are D-amino acids written?
In three-letter notation they carry a D- prefix, as in D-Ala. In one-letter strings lowercase letters are widely used, but the document should define the convention.
Why is the molecular formula on a COA different from the salt form?
The molecular formula usually describes the peptide itself, while counter-ions such as trifluoroacetate are reported as the salt form. Both are needed to relate the weighed mass to the amount of peptide.
What does "pGlu" at the start of a sequence mean?
It indicates an N-terminal pyroglutamate, a five-membered lactam formed from glutamine or glutamic acid. The N-terminal amine is part of the ring, so there is no H- terminus.
Disclaimer. This article is for research and educational purposes only. It describes laboratory and analytical practice and is not medical advice. It contains no dosing, administration or human-use guidance. Veridryx products are sold for laboratory research use only and are not for human or veterinary use.
References
- IUPAC-IUB Joint Commission on Biochemical Nomenclature (JCBN). Nomenclature and Symbolism for Amino Acids and Peptides (Recommendations 1983).
- IUPAC-IUB Commission on Biochemical Nomenclature. A One-Letter Notation for Amino Acid Sequences (Tentative Rules).
- ICH Q6B. Specifications: Test Procedures and Acceptance Criteria for Biotechnological/Biological Products.