Peptide Sequence Notation and End Caps: Reading Ac-, -NH2, cyclo and Salt Forms

Why peptide sequence notation matters on a listing

A sequence string is a specification. Kisspeptin-10, the last ten residues of human kisspeptin, is amidated in nature: UniProt annotates its final residue as “Phenylalanine amide”. Written YNWNSFGLRF-NH2 it is C63H83N17O14, monoisotopic mass 1301.631 Da; the same letters ending -OH give C63H82N16O15 and 1302.615 Da. A certificate that confirms one does not confirm the other.

Databases do not always put caps in the same place. KEGG’s sermorelin record lists 29 residues with no mark at the C-terminus, yet its formula, C149H246N44O42S, is the amide’s (the acid would be C149H245N43O43S), while its thymalfasin record writes “Ac-” into the sequence line. Read the caps in the sequence, then confirm them in the formula or the mass, which cannot leave them out.

Termini: H-, -OH, Ac- and -NH2

IUPAC-IUB builds everything from the hyphen. A hyphen on the left of a residue symbol “signifies removal of a hydrogen atom from the 2-amino” group; one on the right signifies “removal of hydroxyl from the 1-carboxyl group”. Whatever is written beyond the hyphen replaces what was removed. Hence the rule’s own contrast — “Thus H-Ala may be contrasted with Ac-Ala, and Ala-OH with Ala-OMe” — and its amide, “-Ala-NH2 which shows the amide of C-terminal alanine”.

Free ends are usually left unwritten (IUPAC’s own examples include plain Gly-Gly), so a missing mark normally means a free terminus, but only an explicit -OH, a formula or a mass settles it. A cap is part of a molecule’s identity: IUPAC even names the free acid of a natural amide separately, “oxytocinoic acid from oxytocin”. N-terminal pyroglutamate, written Glp in IUPAC’s example for thyroliberin, “Glp-His-Pro-NH2”, and pGlu or pE elsewhere, also blocks the N-terminus; formed from glutamine, it is 17.0265 Da lighter (Unimod).

Rings and bridges: cyclo and disulfide notation

In IUPAC-IUB cyclic peptide notation, the cyclo form is given for homodetic rings, “Cyclic peptides in which the ring consists solely of amino-acid residues in eupeptide linkage”, such as gramicidin S, cyclo(-Val-Orn-Leu-D-Phe-Pro-Val-Orn-Leu-D-Phe-Pro-); by the hyphen rule, the opening and closing hyphens show the last residue bonded back to the first. A ring has no ends, so there is no H- or -OH, and the starting residue is a choice: KEGG starts the same ring at D-Phe, and both describe C60H92N12O10. Head-to-tail closure removes one water, 18.0106 Da.

Rings in which “one or more is an isopeptide, disulfide, ester, or other bond” are heterodetic and are written by the rules for substituted residues. In practice a disulfide is often given as a note naming the paired cysteines, as in UniProt’s feature tables. Arginine vasopressin, Cys-Tyr-Phe-Gln-Asn-Cys-Pro-Arg-Gly-NH2, has a Cys1–Cys6 bridge, which UniProt records at positions 20–25 because it numbers the whole precursor, so check which numbering a document uses. A bridge removes two hydrogens (2.0157 Da): KEGG’s vasopressin formula, C46H65N15O12S2, is two hydrogens short of the open chain. A disulfide-closed peptide labelled cyclo is using the word more loosely than IUPAC-IUB, which gives the cyclo form only for homodetic rings.

Non-standard residues, substituents and labels

D-residues take a D- prefix, “separated from it by a hyphen” in IUPAC’s wording; our guide to how D-residues are written and why covers the stereochemistry. The table decodes the other common marks. IUPAC’s preferred symbol is not always the one on the label.

Isomers hide from mass: norleucine, leucine and isoleucine share a formula, as do D- and L-forms, so only the written sequence records those differences.

Salt forms and other suffixes

Synthetic peptides are often supplied as salts, their basic groups paired with trifluoroacetate (TFA), acetate or chloride. WHO’s guidance says names for different salts of a substance “should differ only with regard to the inactive moiety of the molecule”: sermorelin is the International Nonproprietary Name, and KEGG lists sermorelin acetate separately under its US adopted name.

The suffix names the counter-ion, rarely the amount. KEGG writes pralmorelin dihydrochloride, the salt of GHRP-2, as C45H55N9O6 · 2HCl, molecular weight 890.89 against 817.97 for the peptide alone, with its own CAS number, 158827-34-0; PubChem gives 158861-67-7 for pralmorelin and lists acetate and trifluoroacetate salts among its synonyms. For saralasin acetate, KEGG writes the acetate as (C2H4O2)x and leaves x undefined. The same peptide as a TFA or an acetate salt therefore puts different amounts of peptide in each weighed milligram, although the peptide’s own formula and mass are identical; our guide to net peptide content and TFA versus acetate salts explains how that is measured.

What end caps do: charge, stability and mass

Charge. IUPAC-IUB notes that “The predominant form at pH 7 of a mono-amino-mono-carboxylic acid is R-CH(NH3+)-COO-”; a peptide’s free ends likewise carry a positive amine and a negative carboxylate. Acetylation neutralizes “the positive charge of the N-terminus”, according to a 2018 review that puts the modification on an estimated 80% of human protein species. Amidation removes the negative charge, which the Calgary authors see as the most intuitive reason it raised activity.

Stability. Exopeptidases trim from the ends, aminopeptidases from the N-terminus and carboxypeptidases from the C-terminus, and caps are meant to block them — unevenly. In the Calgary serum study, N-terminal acetylation “confers notable resistance against serum aminopeptidases”, yet “C-terminal amidation does not protect against carboxypeptidase activity in human serum”, apparently because endopeptidases cut near the middle first; protecting both ends gave greater stability. Acetylation also lowered antimicrobial activity while amidation raised it. That is why findings on an unmodified peptide cannot simply be transferred to its capped version: a cap is a structural change whose consequences are measured peptide by peptide. Other ways peptides are modified to last longer, such as lipidation, work on different principles.

Mass. Each change has an exact formula. The values below are Unimod’s or computed from standard atomic masses, and our sibling guide shows how end caps change molecular weight in a full calculation.

The last row is the trap: a peptide that should end in -NH2 but was made as the free acid is 0.9840 Da heavier, exactly the shift of a deamidated asparagine or glutamine, so intact mass alone cannot tell the two apart.

Why so many natural peptide hormones end in an amide

In nature, amidation has a single source. A 2022 review calls peptidylglycine α-amidating monooxygenase (PAM) “the sole enzyme responsible for amidated peptide biosynthesis, from Chlamydomonas reinhardtii to Homo sapiens”, notes that “Many bioactive peptides require a C-terminal α-amide for full activity”, and describes a reaction that “occurs in two steps, glycine α-hydroxylation followed by dealkylation”, leaving the residue before the glycine as an α-amide.

The glycine shows in UniProt’s precursor sequences. The vasopressin, GnRH and thyroliberin precursors each continue Gly-Lys-Arg after the amidated residue, kisspeptin’s continues Gly-Lys-Arg after its final phenylalanine, and the growth hormone-releasing hormone precursor continues Gly-Arg after its final leucine.

Two consequences matter for a catalogue. Sermorelin, GHRH(1–29)-NH2, is amidated at Arg29, although in the natural 44-residue hormone residue 30 is glutamine and the amide sits on Leu44: the fragment carries a cap the natural peptide does not have at that position. Thymosin alpha-1, by contrast, is natively acetylated — UniProt annotates its first residue as N-acetylserine, from processing of prothymosin alpha — and ends as a free acid.

Worked examples from research peptides

Masses are monoisotopic, computed by us from each formula.

• GHRP-2: H-D-Ala-D-2Nal-Ala-Trp-D-Phe-Lys-NH2, C45H55N9O6, 817.428 Da. It has three D-configured residues, one of them non-standard, plus an amide; a matching mass cannot confirm the D-residues.
• Kisspeptin-10: H-Tyr-Asn-Trp-Asn-Ser-Phe-Gly-Leu-Arg-Phe-NH2, C63H83N17O14, 1301.631 Da. The free acid would be 1302.615 Da.
• Sermorelin: H-Tyr-Ala-Asp-…-Ser-Arg-NH2, 29 residues, C149H246N44O42S, 3355.819 Da, matching KEGG’s exact mass.
• Thymosin alpha-1: Ac-Ser-Asp-Ala-…-Glu-Asn-OH, 28 residues, C129H215N33O55, 3106.504 Da, also matching KEGG; without the acetyl group it would be 3064.494 Da.
• Semax (Met-Glu-His-Phe-Pro-Gly-Pro) and Selank (Thr-Lys-Pro-Arg-Pro-Gly-Pro): 813.348 and 751.434 Da. An N-acetyl form adds 42.0106 Da, and a form also sold as amidated a further −0.9840 Da. Our comparison explains what NA and amidate mean on a label. We found no peer-reviewed study comparing either N-acetyl form with its parent, so regard them as distinct reagents.

What’s established and what isn’t

Using notation to check a listing in Canada

Whichever Canadian supplier you use, the checks are the same and need only the listing and certificate side by side. We are a supplier ourselves, so apply them to our listings too.

• Find both ends: Ac- or H- at the start, -NH2 or -OH at the end, confirmed in the molecular formula.
• Decode unusual symbols. D-residues, Orn, Aib, Nal or a cyclo prefix should be defined on the page.
• If the certificate reports a mass, match it to the capped, bridged sequence. A gap of 42.01, 0.98 or 2.02 Da points to a cap or a bridge, though 0.98 Da can also be deamidation.
• Read the salt form. It changes how much peptide a weighed vial holds, not which peptide it is.
• Ask about ambiguous labels, such as NA or amidate without a written structure; our contact page is the route for documentation and product questions.

Pure Peptides Canada is Canadian-owned and based in Toronto; prices are in Canadian dollars, and orders ship tracked from inside Canada.

Frequently asked questions