How to Read a Peptide Sequence: One-Letter Codes, Modifications, and Salt Forms

Research-use-only context. This article summarizes published third-party scientific literature and laboratory practice. It is not medical advice, not a therapeutic or performance claim, and not a usage guide. US Prime Peptides products are supplied strictly for in vitro laboratory research and are not for human or veterinary use.

A peptide sequence is a compact code: letters for residues, a direction convention, prefixes and suffixes for end caps, lowercase or bracketed symbols for unusual residues, and side-chain notes for anything attached. This article shows how to read each part, using the two heptapeptides Semax and Selank as worked examples, and then explains what the sequence does not tell you, including the salt form and the actual mass of peptide in a vial.

The three-letter and one-letter codes

Each of the twenty standard amino acids has a three-letter symbol (Gly, Pro, Glu) and a one-letter symbol (G, P, E), both fixed by the IUPAC-IUB Joint Commission on Biochemical Nomenclature in its 1983 recommendations. The three-letter form is unambiguous and is used on certificates; the one-letter form is compact and is used in databases. Most of the one-letter codes are the initial letter; the exceptions exist because several names share an initial.

Two letters are reserved for ambiguity: B for Asx (Asn or Asp) and Z for Glx (Gln or Glu), used when an analysis cannot tell the amide from the acid. X stands for any residue. These appear in database entries, not on a certificate for a defined synthetic product.

Direction, termini, and end caps

A sequence is always written from the N-terminus (free amine) on the left to the C-terminus (carboxyl group) on the right, and the residues are numbered in that order. Semax is written Met-Glu-His-Phe-Pro-Gly-Pro, or MEHFPGP, and Dolotov and colleagues' 2006 paper in the Journal of Neurochemistry identifies it as an analogue of the 4-10 fragment of adrenocorticotropin with the C-terminal Pro-Gly-Pro added. Selank is Thr-Lys-Pro-Arg-Pro-Gly-Pro, or TKPRPGP; Kolomin and colleagues' 2011 paper in Regulatory Peptides describes it as a synthetic analogue of tuftsin, the natural tetrapeptide TKPR, again extended with Pro-Gly-Pro.

The ends can be capped, and the caps are part of the defined molecule. "H-" at the start and "-OH" at the end mean a free amine and a free acid; they are often omitted because they are the default. "Ac-" at the start means the N-terminal amine carries an acetyl group, which removes its positive charge and adds 42 daltons. Ree, Varland, and Arnesen's 2018 review in Experimental and Molecular Medicine covers acetylation as it occurs in nature; in synthetic peptides it is installed deliberately in the last step on the resin. "-NH2" at the end means the C-terminal carboxyl has been converted to a carboxamide. Merkler's 1994 review of 45 amidated peptides and Eipper, Stoffers, and Mains' 1992 review in the Annual Review of Neuroscience explain why so many natural signalling peptides end this way; in synthesis the amide comes from the choice of resin linker. The product sold as N-acetyl Semax amidate is therefore written Ac-Met-Glu-His-Phe-Pro-Gly-Pro-NH2, a different molecule from Semax with a different mass.

Non-standard residues: D-amino acids and other substitutions

Any residue that is not one of the twenty standard L-amino acids is flagged in the notation. The most common case is a D-amino acid, the mirror-image stereoisomer, which is written with a "D-" prefix in three-letter form (D-Ala) or as a lowercase letter in one-letter form (a). Feng and Xu's 2016 review in Biomolecular Concepts summarizes why D-residues are used: proteases recognize L-residues, so a D-substitution at a cleavage site makes that bond resistant to enzymatic hydrolysis without changing the mass. That last point matters for reading a certificate, since mass spectrometry cannot distinguish a D-residue from its L-isomer; only chiral amino acid analysis or an orthogonal method can.

Other substitutions have their own symbols. Aib is alpha-aminoisobutyric acid, a non-natural residue with two methyl groups that blocks protease cleavage and favors helix formation. Nle is norleucine, a non-oxidizable stand-in for methionine. Dmt is 2',6'-dimethyltyrosine, found in SS-31. Orn is ornithine. Knudsen and Lau's 2019 review in Frontiers in Endocrinology describes NN9535 as human GLP-1(7-37) with two substitutions, Aib at position 8 and arginine at position 34, plus a side-chain attachment at lysine 26; reading that description is simply reading three annotations on a 31-residue sequence.

Side-chain attachments: lipids, PEG, and metals

Anything attached to a side chain is written in parentheses after the residue that carries it, sometimes with the residue number. The NN9535 attachment at lysine 26 is a C18 diacid fatty chain joined through a gamma-glutamate and two short oligo(ethylene glycol) spacers, which a certificate may write as Lys26(gamma-Glu-2xOEG-C18 diacid) or in a longer chemical form. The purpose is albumin binding, as Knudsen and Lau describe, and the attachment roughly doubles the molecule's hydrophobicity, which changes its HPLC retention and its solubility.

PEGylation is written the same way, with the PEG size given in daltons or in ethylene-glycol units, such as Lys(PEG2000) or Cys(PEG24). Harris and Chess's 2003 review in Nature Reviews Drug Discovery explains the chemistry and also why a PEGylated peptide gives a broad mass spectrum instead of a single peak: commercial PEG is a distribution of chain lengths, so the product is a family of masses rather than one. Metal complexes are written as a suffix, as in GHK-Cu, where the tripeptide Gly-His-Lys binds one copper(II) ion; the copper is part of the defined substance and part of its mass.

What the sequence does not tell you

The sequence defines the peptide, but not the solid in the vial. The powder also contains counter-ions, residual water, and traces of salts, none of which appear in the sequence notation. Most synthetic peptides leave the production line as trifluoroacetate salts, because trifluoroacetic acid is used both to cleave the peptide from the resin and as the HPLC mobile-phase modifier; Roux and colleagues' 2008 paper in the Journal of Peptide Science evaluated the methods for exchanging that counter-ion for acetate and found that residual trifluoroacetate should be measured rather than assumed. Cornish and colleagues reported in 1999 that trifluoroacetate at low concentrations altered the proliferation of cultured osteoblasts and chondrocytes, so the salt form is relevant to any cell-based work.

The salt form therefore belongs on the certificate as its own line, next to the sequence. Selank has three basic sites (the N-terminal amine, the lysine, and the arginine), so as a trifluoroacetate salt it carries up to three counter-ions of 113 daltons each; as an acetate salt the same sites carry 59 daltons each. Either way, the mass of peptide is less than the mass of powder, which is why net peptide content is reported separately from purity. The arithmetic is worked through in milligrams, micrograms, and molarity, and the counter-ion question in TFA salt vs acetate salt.

Checking the sequence against the certificate

A certificate confirms the sequence indirectly, by showing that the measured molecular mass matches the mass calculated from the sequence. The calculated value comes from the molecular formula, which PubChem lists for both worked examples; the LC-MS identity line should report an observed mass within a fraction of a dalton of it, after accounting for the charge state and for whether the instrument reports monoisotopic or average mass. How that comparison is made is covered in LC-MS identity confirmation.

Three checks follow from the table. The acetyl cap adds 42 and the amide replaces OH with NH2 (a change of about one dalton), so the capped product should show a mass about 41 daltons above the parent; if the certificate reports the parent mass for a capped product, the identity line and the label disagree. A mass of 16 daltons above expected points to an oxidized methionine, which Semax carries at position 1. And a match in mass says nothing about chirality, so a sequence containing D-residues needs a separate chiral assay. The sequence, formula, salt form, and observed mass are all printed on the lot-matched certificate posted with Semax 10 mg and Selank 10 mg, which is where these checks are meant to be made. For the broader question of why one sequence can carry several names, see why one peptide has many names.

Frequently Asked Questions

How do you read a peptide sequence?

Read it left to right from the N-terminus to the C-terminus, one letter or one three-letter code per residue. A prefix such as Ac- describes a cap on the first amine, a suffix such as -NH2 describes a cap on the last carboxyl, and anything in parentheses after a residue is attached to that residue's side chain.

What does Ac- or -NH2 mean in a peptide sequence?

Ac- means the N-terminal amine carries an acetyl group, which removes its charge and adds 42 daltons. -NH2 means the C-terminal carboxylic acid has been converted to an amide. Both are part of the defined molecule and change its mass, so they should appear in the sequence line of the certificate.

What do lowercase letters in a peptide sequence mean?

A lowercase one-letter code, or a D- prefix in three-letter form, marks a D-amino acid, the mirror image of the standard L-residue. D-residues resist enzymatic cleavage and have the same mass as their L-isomers, so they cannot be confirmed by mass spectrometry alone.

Does the sequence tell you how much peptide is in the vial?

No. The sequence defines the molecule, but the powder also contains counter-ions such as trifluoroacetate or acetate, residual water, and salts. The mass of peptide is given by the net peptide content line on the certificate, and the counter-ion by the salt-form line.

Citations

1. IUPAC-IUB Joint Commission on Biochemical Nomenclature (JCBN). Nomenclature and symbolism for amino acids and peptides. Recommendations 1983. Eur J Biochem. 1984;138:9-37. PMID 6692818
2. Dolotov OV, Karpenko EA, Seredenina TS, et al. Semax, an analogue of adrenocorticotropin (4-10), binds specifically and increases levels of brain-derived neurotrophic factor protein in rat basal forebrain. J Neurochem. 2006;97 Suppl 1:82-86. PMID 16635254
3. Kolomin T, Shadrina M, Andreeva L, Slominsky P, Limborska S, Myasoedov N. Expression of inflammation-related genes in mouse spleen under tuftsin analog Selank. Regul Pept. 2011;170:18-23. PMID 21609736
4. Ree R, Varland S, Arnesen T. Spotlight on protein N-terminal acetylation. Exp Mol Med. 2018;50:1-13. PMID 30054468
5. Merkler DJ. C-terminal amidated peptides: production by the in vitro enzymatic amidation of glycine-extended peptides and the importance of the amide to bioactivity. Enzyme Microb Technol. 1994;16:450-456. PMID 7764886
6. Eipper BA, Stoffers DA, Mains RE. The biosynthesis of neuropeptides: peptide alpha-amidation. Annu Rev Neurosci. 1992;15:57-85. PMID 1575450
7. Feng Z, Xu B. Inspiration from the mirror: D-amino acid containing peptides in biomedical approaches. Biomol Concepts. 2016;7:179-187. PMID 27159920
8. Knudsen LB, Lau J. The Discovery and Development of Liraglutide and NN9535. Front Endocrinol (Lausanne). 2019;10:155. PMID 31031702
9. Harris JM, Chess RB. Effect of pegylation on pharmaceuticals. Nat Rev Drug Discov. 2003;2:214-221. PMID 12612647
10. Roux S, Zekri E, Rousseau B, Paternostre M, Cintrat JC, Fay N. Elimination and exchange of trifluoroacetate counter-ion from cationic peptides: a critical evaluation of different approaches. J Pept Sci. 2008;14:354-359. PMID 18035848
11. Cornish J, Callon KE, Lin CQ, et al. Trifluoroacetate, a contaminant in purified proteins, inhibits proliferation of osteoblasts and chondrocytes. Am J Physiol. 1999;277:E779-E783. PMID 10567002

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