Peptide Sequence Notation: One-Letter Codes, Three-Letter Codes, and Modifications

Peptide sequences are written from the N-terminus (amino end, left) to the C-terminus (carboxyl end, right), following IUPAC-IUB conventions established in 1983. Two parallel notation systems are in standard use: the one-letter code (compact, used for long sequences and databases) and the three-letter code (more readable, used for shorter sequences and when modifications must be indicated clearly).

The choice between systems is largely contextual. Research databases (UniProt, PDB) use one-letter codes. Certificates of Analysis and peptide vendor catalogs typically use three-letter codes for clarity, especially when non-standard amino acids or modifications are present.

The 20 genetically encoded L-amino acids each have an unambiguous one-letter and three-letter designation:

• A = Ala (Alanine)
• C = Cys (Cysteine)
• D = Asp (Aspartic acid)
• E = Glu (Glutamic acid)
• F = Phe (Phenylalanine)
• G = Gly (Glycine)
• H = His (Histidine)
• I = Ile (Isoleucine)
• K = Lys (Lysine)
• L = Leu (Leucine)
• M = Met (Methionine)
• N = Asn (Asparagine)
• P = Pro (Proline)
• Q = Gln (Glutamine)
• R = Arg (Arginine)
• S = Ser (Serine)
• T = Thr (Threonine)
• V = Val (Valine)
• W = Trp (Tryptophan)
• Y = Tyr (Tyrosine)

• B = Asx (Asparagine or Aspartic acid — ambiguous)
• Z = Glx (Glutamine or Glutamic acid — ambiguous)
• J = Xle (Leucine or Isoleucine — ambiguous, used in MS sequencing where isobaric residues cannot be distinguished)
• X = Xaa (Any amino acid or unknown residue)
• U = Sec (Selenocysteine — the 21st amino acid)
• O = Pyl (Pyrrolysine — the 22nd amino acid)

• H- (free amine): The default, unmodified N-terminus. Often omitted in notation: H-ACDEFG-OH is equivalent to ACDEFG-OH.
• Ac- (acetyl): Acetylation of the N-terminal amine. Adds +42.04 Da. Written as Ac-SEQUENCE-OH. Blocks the positive charge and increases metabolic stability.
• Fmoc- (fluorenylmethyloxycarbonyl): Synthesis protecting group. Should NOT be present on a finished peptide — its presence indicates incomplete deprotection.
• pGlu- or pE- (pyroglutamate): Cyclized glutamic acid or glutamine at the N-terminus. Occurs naturally and may form spontaneously from N-terminal Gln.
• Biotin- : Biotinylation for streptavidin binding applications. Adds +226.29 Da.
• FITC- or FAM-: Fluorescent labels (fluorescein isothiocyanate or carboxyfluorescein).

• -OH (free acid): The default, unmodified C-terminus with a free carboxyl group. Standard for most synthetic peptides.
• -NH₂ (amide): Amidation of the C-terminal carboxyl. Removes the negative charge, increases stability, and is common in naturally occurring bioactive peptides. Mass difference: −1.00 Da compared to the free acid form.
• -OMe (methyl ester): Methylation of the C-terminal carboxyl. Rare in research peptides.
• The C-terminal modification significantly affects the molecular weight and must be specified on the CoA.

D-enantiomers of standard amino acids are denoted by lowercase letters in one-letter code (e.g., a = D-Ala, f = D-Phe) or by the prefix D- or (D) in three-letter code (e.g., D-Ala, (D)Phe). D-amino acids increase resistance to proteolytic degradation and are commonly used in synthetic peptide design.

• Aib = α-aminoisobutyric acid (α-methylalanine) — helix stabilizer
• Orn = Ornithine — Arg analog without guanidinium, +1 charge at physiological pH
• Nle = Norleucine — Met isostere, resistant to oxidation
• Cit = Citrulline — Arg analog without charge
• Sar = Sarcosine (N-methylglycine) — N-methylated Gly
• β-Ala = β-alanine — spacer residue with extended backbone
• GABA = γ-aminobutyric acid — spacer with even longer backbone
• Dab = 2,4-diaminobutyric acid — shorter Lys analog
• Dap = 2,3-diaminopropionic acid — shortest diamine residue
• Hyp = Hydroxyproline — hydroxylated Pro, found in collagen

Modifications are indicated in parentheses after the affected residue or as prefixes/suffixes:

• Phosphorylation: pS, pT, pY or S(PO₃H₂), T(PO₃H₂), Y(PO₃H₂) — adds +79.97 Da
• Methylation: K(Me), K(Me₂), K(Me₃) — mono/di/trimethylation of Lys
• Acetylation (side chain): K(Ac) — acetylation of Lys ε-amine
• PEGylation: K(PEG₂₀₀₀) — attachment of polyethylene glycol chain
• Cyclization: Cyclo(sequence) — head-to-tail cyclization
• Disulfide: Cys₁-Cys₅ or indicated by connectivity notation
• Stapling: S₅ or R₈ — hydrocarbon-stapled residues for helix stabilization
• Fatty acid conjugation: Palm-K or Myr-K — palmitoylation or myristoylation

A CoA might list: Ac-Gly-His-Lys-D-Phe-Ala-Glu-NH₂. This notation tells us:

• Ac- → N-terminus is acetylated
• Gly-His-Lys-D-Phe-Ala-Glu → six residues, with the fourth being D-phenylalanine (non-natural enantiomer)
• -NH₂ → C-terminus is amidated
• Expected MW calculation: sum all residue masses + Ac (+42.04) − H₂O (−18.02, peptide bond water loss per bond) + NH₂ amide adjustment (−0.98 vs free acid)

• Count the residues and verify against the stated sequence length
• Identify any non-standard residues or modifications and confirm their mass contributions
• Calculate the expected molecular weight independently and compare to the CoA value
• Check that terminal modifications are correctly reflected in the stated MW
• Verify that disulfide bonds (if any) are accounted for in the mass (−2.016 Da per bond)
• Confirm the sequence notation is internally consistent — the same sequence should produce the same MW regardless of whether one-letter or three-letter code is used

One-letter codes (e.g., A for alanine, G for glycine) are compact and used in sequence alignments, database entries, and bioinformatics tools. Three-letter codes (e.g., Ala, Gly) are more readable and are standard in synthesis orders, chemical literature, and when specifying modifications. Both follow N→C terminal direction.

D-amino acids are denoted with a lowercase 'd' prefix in three-letter notation (e.g., d-Ala, d-Phe) or with a lowercase letter in certain one-letter conventions. In some systems, D-amino acids use italic 'D-' prefix. Always verify the convention used by your supplier or publication.

N-terminal modifications are written before the first residue (e.g., Ac-Ala for N-acetylation, FITC-Gly for fluorescein labeling). C-terminal modifications follow the last residue (e.g., Ala-NH2 for amidation). These modifications are critical for peptide stability and should always be explicitly specified in orders.

Cyclic peptides are indicated by 'cyclo()' wrapping the sequence (e.g., cyclo(Arg-Gly-Asp-d-Phe-Lys)). Side-chain-to-side-chain cyclizations specify the linkage type and residue positions. Disulfide-cyclized peptides show the Cys residues with a connecting notation (e.g., Cys¹-Cys⁶).

Non-natural amino acids use systematic three-letter abbreviations in brackets or parentheses within the sequence (e.g., Aib for α-aminoisobutyric acid, Nle for norleucine, β-Ala for beta-alanine). PEG spacers and other non-amino acid linkers are written as chemical names or abbreviations (e.g., Ahx for aminohexanoic acid).

• Read more: Peptide Aggregation: Why Peptides Clump and How to Prevent It → https://www.chemverify.com/learn/peptide-aggregation-clumping-prevention
• Read more: Peptide Degradation: Deamidation, Oxidation, and How to Prevent It → https://www.chemverify.com/learn/peptide-degradation-deamidation-oxidation-prevention
• Read more: Amino Acid Reference Table: Properties, Structures, and Classification → https://www.chemverify.com/learn/amino-acid-reference-table
• Read more: Peptide Modifications: PEGylation, Lipidation, Cyclization, and D-Amino Acids → https://www.chemverify.com/learn/peptide-modifications-pegylation-lipidation-cyclization