Peptide Sequence Notation: One-Letter and Three-Letter Codes, Termini and Modified Residues

Peptide sequence notation writes a chain of amino-acid residues from the N-terminus on the left to the C-terminus on the right, in either three-letter codes (Gly-Glu-Pro) or one-letter codes (GEP). Prefixes and suffixes state the terminal groups: H- and -OH mark a free amine and a free acid, Ac- an acetylated N-terminus, -NH2 a C-terminal primary amide. A D- prefix or a lower-case letter marks a D-configured residue, brackets with "cyclo" mark a ring, and named abbreviations such as Dmt, Aib or Nle mark residues outside the twenty standard amino acids. Each element of the notation changes the molecular formula and mass in a fixed, calculable way, which is why the notation on a listing can be checked against the mass on an analytical report.

Which direction is a peptide sequence written?

A peptide sequence is always written from the N-terminus (the end with the free amino group) on the left to the C-terminus (the end with the carboxyl group) on the right. Reversing the order describes a different molecule with the same formula and the same mass. BPC-157 is written H-Gly-Glu-Pro-Pro-Pro-Gly-Lys-Pro-Ala-Asp-Asp-Ala-Gly-Leu-Val-OH, or GEPPPGKPADDAGLV in one-letter code: glycine is residue 1 at the N-terminus and valine is residue 15 at the C-terminus. That sequence corresponds to the formula C62H98N16O22 and the 1419.53 g/mol recorded for BPC-157 (PubChem CID 9941957, CAS 137525-51-0).

Residues are numbered from the N-terminus, so "Asp2" or "Lys7" means the aspartic acid at position 2 or the lysine at position 7. Each hyphen between two residue codes in the three-letter form stands for a peptide bond, and every peptide bond forms with the loss of one water molecule (H2O, 18.02 Da average), so a peptide of n residues weighs the sum of its free amino acids minus (n - 1) waters.

What do the one-letter and three-letter amino acid codes mean?

The one-letter and three-letter codes are two abbreviations for the same twenty standard amino acids. The three-letter form (Gly-Glu-Pro) is easier to read and leaves room for modified residues; the one-letter form (GEP) is used for long sequences, such as the 43 residues of TB-500 or the 46 residues of FOXO4-DRI. Several one-letter codes are not the initial of the name: D is aspartic acid, E glutamic acid, N asparagine, Q glutamine, K lysine, R arginine, F phenylalanine, W tryptophan and Y tyrosine.

Short peptides are often named after their own one-letter sequence. KPV is Lys-Pro-Val, written H-Lys-Pro-Val-OH, with the formula C16H30N4O4 and 342.44 Da on the Pepta Labs listing (PubChem CID 125672, CAS 67727-97-3). The tetrapeptide KEDA follows the same convention: Livagen is Lys-Glu-Asp-Ala, covered in the Livagen (KEDA) reference. The Epithalon listing records its own formula and molecular weight.

A residue mass is the mass of an amino acid inside a chain: the free amino acid minus one H2O. The values above are computed from each residue formula with the same atomic masses the site uses for its expected-mass table. Leucine and isoleucine share the residue formula C6H11NO and the same mass, so an intact-mass measurement cannot tell them apart.

What do H-, -OH, Ac- and -NH2 mean at the ends of a sequence?

Prefixes and suffixes attached with a hyphen describe the two terminal groups, not additional residues. H- at the start means the N-terminal amine is free (the H is a hydrogen atom, not histidine), and -OH at the end means the C-terminal carboxylic acid is free. BPC-157, H-Gly-...-Val-OH, has both termini unmodified. Many catalogues omit H- and -OH, so a sequence written without terminal marks is normally read as a free amine and a free acid.

Ac- means the N-terminal amine carries an acetyl group, which adds C2H2O to the formula: +42.01 Da monoisotopic, +42.04 Da average. TB-500 on the Pepta Labs listing is Ac-Ser-Asp-Lys-...-Gly-Glu-Ser-OH: the 43-residue chain without the acetyl group computes to C210H348N56O77S, and with it to C212H350N56O78S, the formula on the listing (4963.44 Da; PubChem CID 16132341, UNII 2D5MRE3SSY).

-NH2 means the C-terminus is a primary amide instead of a free carboxylic acid, which replaces OH with NH2 and lowers the mass by 0.98 Da. SS-31 is written H-D-Arg-Dmt-Lys-Phe-NH2, with the formula C32H49N9O5 and 639.78 g/mol (PubChem CID 11764719, CAS 736992-21-5); the same four residues ending in a free acid would compute to C32H48N8O6. SNAP-8 carries both terminal marks on one chain: Ac-Glu-Glu-Met-Gln-Arg-Arg-Ala-Asp-NH2, or Ac-EEMQRRAD-NH2 in one-letter form, where the Ac- and -NH2 stay in place around the one-letter string. The SS-31 buying guide covers the two SS-31 listings and their reports.

An N-terminal cap other than acetyl is written out by name. Tesamorelin, (3E)-Hex-3-enoyl-Tyr-Ala-Asp-...-Arg-Leu-NH2, is a 44-residue amide whose first 29 residues match sermorelin (Tyr-Ala-Asp-...-Ser-Arg-NH2, C149H246N44O42S, 3357.93 Da); the hexenoyl cap adds C6H8O, +96.06 Da monoisotopic, and the listed formula is C221H366N72O67S at 5135.86 Da (CAS 218949-48-5). The CJC-1295 no DAC, sermorelin and tesamorelin reference sets the three sequences side by side.

What do N-acetyl and amidate mean in a peptide name?

"Amidate" in a peptide product name means the C-terminus is a primary amide (-NH2 in the sequence), and "N-acetyl" means the N-terminus carries an acetyl group (Ac-). Chemists usually write "amide"; "amidate" is the form used in vendor product names. The residue sequence does not change, so N-Acetyl Semax Amidate is Ac-Met-Glu-His-Phe-Pro-Gly-Pro-NH2, the seven residues of Semax (H-Met-Glu-His-Phe-Pro-Gly-Pro-OH) with both termini modified. The two changes together add C2H3N, about 41 Da, to the parent formula.

Because the acetyl and amide groups are covalent, the acetylated, amidated form is a distinct compound with its own formula and mass, not a salt or a grade of the parent peptide (Semax, PubChem CID 9811102; Selank, PubChem CID 11765600). The Semax and Selank buying guide covers the four listings, sizes and reports.

Is an N-acetyl group the same as an acetate salt?

No: an acetyl group is covalently bonded to the peptide and is part of its formula, while acetate is a counter-ion in the salt and is not. "N-acetyl selank amidate acetate" therefore names a peptide with an acetylated N-terminus and an amidated C-terminus, isolated as its acetate salt. The formula and molecular weight on a listing are those of the peptide; the counter-ion and residual water add to the solid mass in the vial, and net peptide content is not reported per listing. See TFA vs acetate salts and net peptide content.

What does a D- prefix or a lower-case letter mean?

A D- prefix in three-letter notation, or a lower-case letter in one-letter notation, marks a residue with the D-configuration at its alpha carbon instead of the usual L-configuration. Residues written without a prefix are L. A D-residue has the same formula and the same mass as its L-form, so the notation records configuration only. SS-31 begins with D-Arg: H-D-Arg-Dmt-Lys-Phe-NH2. Ipamorelin, H-Aib-His-D-2-Nal-D-Phe-Lys-NH2, carries two D-residues at positions 3 and 4; see the ipamorelin buying guide. Glycine has no stereocentre and never carries a D- or L- prefix.

Isoleucine and threonine carry a second stereocentre in the side chain: D-Ile and D-Thr name the full mirror images of the L-forms, and a residue inverted at only one of the two centres is an allo form, as in D-allo-Ile.

An intact-mass result from mass spectrometry cannot distinguish D- from L-residues, or one order of the same residues from another: it confirms the formula. Telling such isomers apart takes additional methods, such as fragmentation (MS/MS) sequencing or chiral chromatography; the testing Pepta Labs represents is RP-HPLC purity and mass-spectrometry identity.

What does D-retro-inverso mean, shown on the FOXO4-DRI sequence?

A D-retro-inverso (DRI) peptide is built from D-amino acids placed in the reverse order of a parent all-L sequence, so it has the same residues, the same formula and the same mass as the parent but the opposite configuration and the opposite chain direction. "Retro" names the reversed residue order and "inverso" the inverted configuration at every chiral residue. Reversing the order and inverting every stereocentre together leave the side chains in approximately the same relative arrangement as in the parent chain, while every backbone amide bond (-CO-NH-) points the other way along the chain.

FOXO4-DRI is the one D-retro-inverso peptide in the Pepta Labs catalog and, at 46 residues, the longest sequence recorded for any listing. The FOXO4-DRI listing writes it as H-LTLRKEPASEIAQSILEAYSQNGWANRRSGGKRPPPRRRQRRKKRG-OH with the note "chiral residues D-configuration; Gly achiral". It runs from the N-terminal D-leucine (residue 1) to the C-terminal glycine (residue 46), with a free amine and a free acid. The listed formula is C228H388N86O64 and the molecular weight 5358.15 g/mol (PubChem CID 167312269, CAS 2460055-10-9). Summing the 46 residues plus one water gives the same C228H388N86O64 (calculated).

The same chain can be written three ways:

• One-letter, upper case with a note (the listing's form): LTLRKEPASEIAQSILEAYSQNGWANRRSGGKRPPPRRRQRRKKRG, chiral residues D.
• One-letter, lower case for D: ltlrkepaseiaqsileaysqnGwanrrsGGkrppprrrqrrkkrG, with the four glycines left upper case because glycine has no D-form.
• Three-letter with D- prefixes: H-D-Leu-D-Thr-D-Leu-D-Arg-D-Lys-D-Glu-D-Pro-D-Ala-D-Ser-D-Glu-...-D-Arg-D-Arg-D-Lys-D-Lys-D-Arg-Gly-OH.

How is the FOXO4-DRI sequence related to its parent sequence?

Residue i of the listed D-retro-inverso chain corresponds to residue 47 - i of the all-L parent chain, so reading the listed string backwards gives the parent sequence in the usual N-to-C direction. Reversed, the listed string reads GRKKRRQRRRPPPRKGGSRRNAWGNQSYAELISQAIESAPEKRLTL (calculated). The table maps each block of ten.

Counted from the listed sequence, the 46 residues are 10 Arg; 4 each of Lys, Pro, Ala, Ser and Gly; 3 each of Leu, Glu and Gln; 2 each of Ile and Asn; and 1 each of Thr, Tyr and Trp. Without the 4 glycines, 42 residues are D. Because both ends are free (H- and -OH), reversing the chain changes no atoms: the parent and the D-retro-inverso chain share the formula exactly.

Which sequences share the FOXO4-DRI formula?

Four chains built from the same 46 residues share the formula C228H388N86O64 and the mass 5358.15 g/mol: the all-L parent, its all-L reversed (retro) form, its all-D (inverso) form and the D-retro-inverso form that is listed.

The listed chain and the all-L retro chain are mirror images, so they give the same mass and the same retention time on an achiral RP-HPLC column. Order differences show in MS/MS fragment ions; configuration differences need a chiral method. The listing records CAS 2460055-10-9 and PubChem CID 167312269; because the four forms share one formula and one mass, a registry record is matched to a listing by its sequence and configuration, not by formula or mass; peptide registry discrepancies explains how to compare a listing with a registry record.

What masses should a FOXO4-DRI mass spectrum show?

For C228H388N86O64 the calculated monoisotopic mass is 5354.98 Da and the calculated average mass 5358.15 Da, 3.17 Da apart. A chain carrying 10 arginines, 4 lysines and a free N-terminal amine has 15 basic sites, so an electrospray spectrum is expected to show a series of multiply charged ions rather than [M+H]+ alone. The values below are calculated from the formula with a proton mass of 1.007276 Da; for a chain of this size a low-resolution centroid is compared with the average column.

The 15 basic sites also mean FOXO4-DRI is normally isolated as a multi-trifluoroacetate or acetate salt; the counter-ions are not part of the formula or of these ion masses.

What are Dmt, Aib, Nle and pGlu in a peptide sequence?

Dmt, Aib, Nle and pGlu are abbreviations for residues outside the twenty standard amino acids, and each has a fixed formula relative to a standard residue.

• Dmt is 2',6'-dimethyl-L-tyrosine: tyrosine with two methyl groups on the aromatic ring, C2H4 heavier than Tyr (+28.03 Da monoisotopic). It is residue 2 of SS-31, which is why the SS-31 formula differs from that of Arg-Tyr-Lys-Phe-NH2 (C30H45N9O5) by C2H4. Dmt has no one-letter code, so SS-31 is written in three-letter form.
• Aib is 2-aminoisobutyric acid (2-methylalanine), residue formula C4H7NO, one CH2 heavier than alanine. It is the N-terminal residue of ipamorelin, H-Aib-His-D-2-Nal-D-Phe-Lys-NH2 (C38H49N9O5, 711.85 g/mol, PubChem CID 9831659), whose position 3 is a second non-standard residue, D-2-Nal, 3-(2-naphthyl)-D-alanine.
• Nle is norleucine, a straight-chain isomer of leucine with the same residue formula (C6H11NO) and the same mass. It appears as Ac-Nle at the N-terminus of Melanotan-II and PT-141.
• pGlu (also written pE or Pyr) is pyroglutamate: an N-terminal residue whose side chain has cyclized onto its own amine to form a lactam, so the peptide has no free N-terminal amine. It is 18.01 Da lighter than Glu (loss of H2O) or 17.03 Da lighter than Gln (loss of NH3). Thymulin is written pGlu-Ala-Lys-Ser-Gln-Gly-Gly-Ser-Asn-OH (C33H54N12O15, 858.86 g/mol).

How is a cyclic peptide written?

A ring is written with "cyclo" and brackets around the residues it contains, and each ring-closing amide bond removes one H2O (18.01 Da) from the formula. The Melanotan-II listing records its sequence as Ac-Nle-cyclo[Asp-His-D-Phe-Arg-Trp-Lys]-NH2, with the lactam between the Asp2 side-chain carboxyl and the Lys7 side-chain amine. The bracket closes the ring from Asp2 to Lys7; Ac-Nle sits outside it at the N-terminus and -NH2 marks the C-terminal amide. The open-chain form of the same residues computes to C50H71N15O10, and the lactam brings it to C50H69N15O9 and 1024.18 Da, the listed values (CAS 121062-08-6).

PT-141 is written Ac-Nle-cyclo[Asp-His-D-Phe-Arg-Trp-Lys]-OH: the same cap, the same ring and the same seven residues, with a free acid in place of the amide. That one suffix is the whole difference: C50H68N14O10 at 1025.17 Da against C50H69N15O9 at 1024.18 Da, and the two listings carry separate CAS numbers (189691-06-3 and 121062-08-6). See the Melanotan-II and PT-141 buying guides. A disulfide ring between two cysteines is written with the same bracket style or as a note such as "disulfide Cys7-Cys14", and removes two hydrogens (2.02 Da) instead of a water.

How does each notation element change a peptide's mass?

Mass changes are monoisotopic and calculated from the formula change. The average-mass equivalents differ slightly: +42.04 for Ac-, +96.13 for the hexenoyl cap, +28.05 for Dmt and -18.02 for one water. Formulas and molecular weights for named compounds are those recorded on each Pepta Labs product listing.

How does residue count relate to molecular weight?

A common rule of thumb is about 110 Da per residue, but for short or unusual sequences it is only an order-of-magnitude estimate; the exact value comes from the formula. Dividing each listed molecular weight by its residue count shows the spread:

BPC-157 falls below the estimate because 9 of its 15 residues are glycine, alanine or proline, each lighter than 100 Da as a residue. SS-31 falls above it because three of its four residues (Arg, Dmt and Phe) are heavier than 145 Da, and FOXO4-DRI sits above it because 10 of its 46 residues are arginine at 156.19 Da each. To check a report, compute the mass from the formula, or look it up in the CAS numbers and molecular weights reference.

How do you check a sequence against an analytical report?

Compute the expected mass from the notation, then compare it with the observed mass in the mass-spectrometry result; the RP-HPLC result reports purity as the main-peak area percentage, not identity. The testing Pepta Labs represents is "Independent third-party HPLC purity analysis and mass-spectrometry identity confirmation, commissioned through the purchasing group on a sampled production run from the source. Pepta Labs does not test in-house." Reports are "Sent by email on request, before you order if you want to see it first." Vial labels "do not carry lot or batch numbers", so a report covers the sampled production run rather than an individual vial. See mass spectrometry for peptide identity and how to review an analytical report.

Frequently asked questions

What is the difference between one-letter and three-letter peptide codes?

One-letter and three-letter codes are two abbreviations for the same residues. GEP and Gly-Glu-Pro both read glycine, glutamic acid, proline from the N-terminus; the one-letter form is used when a sequence is long, such as the 46-residue FOXO4-DRI, and the three-letter form when it contains non-standard residues such as Dmt that have no one-letter code.

What does Ac- at the start of a peptide sequence mean?

Ac- at the start of a peptide sequence means the N-terminal amine carries an acetyl group, which adds C2H2O (42.01 Da monoisotopic) to the formula and mass relative to the free amine, as in TB-500 (C212H350N56O78S, 4963.44 Da).

What does -NH2 at the end of a peptide sequence mean?

-NH2 at the end of a peptide sequence means the C-terminus is a primary amide instead of a free carboxylic acid. OH is replaced by NH2, which lowers the mass by 0.98 Da, as in SS-31 (H-D-Arg-Dmt-Lys-Phe-NH2) and SNAP-8 (Ac-EEMQRRAD-NH2); Melanotan-II (-NH2, 1024.18 Da) and PT-141 (-OH, 1025.17 Da) differ only in this suffix.

What does amidate mean in a peptide name?

"Amidate" in a peptide name means the C-terminus is a primary amide, written -NH2 at the end of the sequence. With "N-acetyl" (Ac-) the two terminal changes add C2H3N, about 41 Da, to the parent formula: Semax (C37H51N9O10S, 813.90 Da) becomes N-Acetyl Semax Amidate (C39H54N10O10S, 854.98 Da). See the Semax and Selank buying guide for the listings.

Does a D-amino acid change the molecular weight?

No: a D-amino acid has the same formula and mass as its L-form. D-Arg in SS-31 has the same formula and mass as L-Arg; the notation records configuration only, and an intact-mass measurement cannot tell the two apart. Even FOXO4-DRI, with 42 D-residues, has the same formula (C228H388N86O64) and mass (5358.15 g/mol) as its all-L parent.

What does D-retro-inverso mean?

D-retro-inverso means a peptide is built from D-amino acids in the reverse order of a parent all-L sequence, so the formula and mass do not change. In the 46-residue FOXO4-DRI (C228H388N86O64, 5358.15 g/mol, CAS 2460055-10-9), residue i of the listed chain matches residue 47 - i of the parent.

Sources

• PubChem CIDs 9941957 (BPC-157), 16132341 (TB-500), 11764719 (SS-31), 125672 (KPV), 167312269 (FOXO4-DRI), 9811102 (Semax), 11765600 (Selank), 16132413 (sermorelin), 16137828 (tesamorelin), 92432 (Melanotan-II), 9941379 (PT-141), 9831659 (ipamorelin) and 71300623 (thymulin).
• CAS Registry Numbers as recorded in PubChem: 137525-51-0 (BPC-157), 736992-21-5 (SS-31), 67727-97-3 (KPV), 2460055-10-9 (FOXO4-DRI), 218949-48-5 (tesamorelin), 121062-08-6 (Melanotan-II) and 189691-06-3 (PT-141).
• FDA Global Substance Registration System UNIIs 8ED8NXK95P (BPC-157), 2D5MRE3SSY (TB-500), 87GWG91S09 (SS-31), Y9M3S784Z6 (ipamorelin), MQG94M5EEO (tesamorelin), UPF5CJ93X7 (Melanotan-II) and 6Y24O4F92S (PT-141).
• Pepta Labs product listings for sequences, formulas and listed molecular weights. Residue masses, formula changes, the reversed FOXO4-DRI sequence and the FOXO4-DRI ion table are calculated from formulas and sequences.

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