A peptide sequence is an ordered list of amino-acid residues, written in one of two codes and read from the N-terminus to the C-terminus. This covers both codes, reading direction and numbering, modified chain ends, cyclic and disulfide notation, residues outside the standard twenty, and two catalog sequences read field by field.
One-letter and three-letter codes
Two codes name the same twenty standard residues. The three-letter code contracts each residue name to its first three letters and hyphenates them: Gly-His-Lys. The one-letter code gives each residue a single capital and runs them together: GHK. Both are set by the IUPAC-IUB Joint Commission on Biochemical Nomenclature, and either can write any sequence built only from the twenty.
A residue is what is left of an amino acid once it is joined into a chain: the free amino acid less the water lost in forming the peptide bond, and so the lighter of the two.
Eleven residues take their own initial; the other nine share one with a residue that took it first and carry an associated letter instead.
| Residue | Three-letter | One-letter |
|---|---|---|
| Alanine | Ala | A |
| Arginine | Arg | R |
| Asparagine | Asn | N |
| Aspartic acid | Asp | D |
| Cysteine | Cys | C |
| Glutamic acid | Glu | E |
| Glutamine | Gln | Q |
| Glycine | Gly | G |
| Histidine | His | H |
| Isoleucine | Ile | I |
| Leucine | Leu | L |
| Lysine | Lys | K |
| Methionine | Met | M |
| Phenylalanine | Phe | F |
| Proline | Pro | P |
| Serine | Ser | S |
| Threonine | Thr | T |
| Tryptophan | Trp | W |
| Tyrosine | Tyr | Y |
| Valine | Val | V |
The one-letter code is compact and is what database records and search fields take. A residue from outside the twenty generally has to be written in the three-letter code, selenocysteine and pyrrolysine being the exceptions that carry letters of their own.
Both codes distinguish residues that a mass measurement does not. Leucine and isoleucine are structural isomers, both C₆H₁₁NO as residues, with the same monoisotopic residue mass of 113.084 Da. What the instruments resolve is in peptide purity by RP-HPLC and ESI-MS.
Reading direction
A peptide sequence is written left to right from the N-terminus to the C-terminus. The N-terminus is the chain end carrying a free α-amino group; the C-terminus is the end carrying a free α-carboxyl group. A peptide has one of each unless an end has been modified or the backbone has been closed into a ring.
The convention matches the direction the chain is built biologically: a ribosome adds the N-terminal residue first and extends towards the C-terminus. Chemical synthesis runs the other way: solid-phase synthesis anchors the C-terminal residue to the resin and couples each new residue onto the free amine, so a chain is built in the reverse of the order it is written.
Numbering runs over residues only, from 1 at the N-terminus, so an N-terminal acetyl group, a C-terminal amide and a bound metal ion take no number. A range in a compound name counts residues of a named parent sequence: the catalog’s Sermorelin, GHRH (1–29), covers residues 1 through 29 of that parent counted from its N-terminus.
Order carries information that composition does not: TKPRPGP and PGPRPKT hold the same seven residues and weigh the same, and are different molecules.
Terminal modifications
Two modifications of the chain ends have fixed notation. N-terminal acetylation replaces one hydrogen of the free α-amino group with an acetyl group and is written as the prefix Ac- before the first residue. C-terminal amidation replaces the hydroxyl of the free α-carboxyl with an amide and is written as the suffix -NH₂ after the last residue.
An unmodified end may be stated explicitly, H- for a free α-amino group and -OH for a free α-carboxyl, and is otherwise understood by omission: Ac-LKKTETQ declares an acetylated N-terminus and, by silence, a free acid C-terminus. Each modification shifts the mass by a fixed amount.
| Notation | End | Records | Mass shift |
|---|---|---|---|
| Ac- | N-terminus | Acetyl on the α-amino group | +42.011 Da |
| H- | N-terminus | Free α-amino group; the default | 0 |
| pGlu- | N-terminus | N-terminal Gln or Glu cyclised to pyroglutamate | −17.027 Da from Gln, −18.011 Da from Glu |
| -NH₂ | C-terminus | α-carboxamide | −0.984 Da |
| -OH | C-terminus | Free α-carboxyl; the default | 0 |
A C-terminal amide is 0.984 Da lighter than the corresponding free acid. Overlook the -NH₂ and the calculated mass comes out 0.984 Da high. Deamidation of an asparagine or glutamine side chain moves the mass the same amount the other way. How a difference that size is read on a certificate is in how to read a Certificate of Analysis.
Cyclic peptides and disulfide bonds
A ring has no chain end to start reading from, so the notation states where it closes. A head-to-tail cyclic peptide, whose C-terminus is joined to its own N-terminus through an ordinary peptide bond, is written with the whole residue list inside cyclo( ): cyclo(-Gly-Pro-Phe-Ala-). The residue written first is a starting point, not a terminus; the ring may be listed from any residue as long as the order is kept.
A side-chain bridge closes a smaller ring inside an otherwise linear chain, most often a lactam between an aspartate or glutamate carboxyl and a lysine amine. The bracket encloses only the span between the two joined residues: Ac-Ala-cyclo(Asp-Gly-Phe-Lys)-NH₂.
A disulfide bond links the sulfur atoms of two cysteine residues. It is no part of the backbone, so it is written as the pair of residue numbers it joins, Cys1-Cys6 or 1–6, beside the sequence, or drawn as a line above the chain. Two or more bridges are given as a list of pairs, and the pairing matters: the same four cysteines can be joined in more than one arrangement.
| Notation | Reads as |
|---|---|
| cyclo(-Gly-Pro-Phe-Ala-) | Head-to-tail ring; backbone closed C-terminus to N-terminus, no free ends |
| Ac-Ala-cyclo(Asp-Gly-Phe-Lys)-NH₂ | Side-chain bridge across the enclosed span; the rest of the chain is linear |
| Cys1-Cys6 | One disulfide, between the residues numbered 1 and 6 |
| 1–6, 2–7 | Two disulfides, in that pairing and not the alternative one |
Forming a disulfide gives up two hydrogen atoms, so a bridged chain is 2.016 Da lighter than the same chain with free thiols, per bridge. A sequence naming two cysteines but no pairing has not said whether the bond is formed.
Non-standard residues
Most residues outside the twenty standard ones have no one-letter code, so a sequence containing one is generally written in the three-letter code. Selenocysteine and pyrrolysine are the exceptions, carrying the letters U and O.
The nineteen chiral standard residues are L-enantiomers, so L is the default and is left unwritten and a D-residue is marked: D-Phe, D-Ala. Glycine is the exception: its α-carbon carries two hydrogens, so it is achiral and has neither an L nor a D form. Some software lower-cases the one-letter code instead, f for D-Phe, a local convention rather than an IUPAC one. A D-residue has the same formula and mass as its L-form, so a mass measurement cannot see the substitution.
Aib is α-aminoisobutyric acid, glycine carrying two methyl groups on the α-carbon, monoisotopic residue mass 85.053 Da. Those two identical substituents leave it achiral for the same reason glycine is, so it takes no L or D. It has no letter and is written Aib. Others written the same way include Nle norleucine, Orn ornithine, Sar sarcosine, Cit citrulline and Hyp hydroxyproline.
Six letters stand for something other than one of the twenty, and four of the six name no residue at all: X leaves one unspecified, and B, Z and J record an ambiguous determination. Only U and O are residues.
| Letter | Three-letter | Stands for |
|---|---|---|
| X | Xaa | Any residue, or one left unspecified |
| B | Asx | Asp or Asn, not distinguished by the determination |
| Z | Glx | Glu or Gln, not distinguished by the determination |
| J | Xle | Leu or Ile, not distinguished by the determination |
| U | Sec | Selenocysteine, a genetically encoded residue outside the twenty |
| O | Pyl | Pyrrolysine, a genetically encoded residue outside the twenty |
The ambiguity letters name the method’s limit. Acid hydrolysis converts the side-chain amides of asparagine and glutamine to the corresponding acids, so a composition determined that way cannot separate Asp from Asn or Glu from Gln: B and Z record that. J records the same for the isobaric leucine and isoleucine. A catalog or certificate sequence should carry none of the three.
Worked examples
Two catalog sequences follow, one in each code. Reading either means identifying the residues, finding the ends, and accounting for anything printed that is not a residue.
GHK-Cu, Copper Tripeptide-1, CAS 89030-95-5, is listed with the sequence Gly·His·Lys, and the header block of Vitro’s certificate shows Gly-His-Lys · Cu²⁺.
| Printed | Reads as |
|---|---|
| Gly·His·Lys | Three residues in the three-letter code: glycine, histidine, lysine, in that order |
| Gly | Residue 1, the N-terminus, carrying a free α-amino group |
| Lys | Residue 3, the C-terminus, carrying a free α-carboxyl |
| · in the catalog, - on Vitro’s certificate | Separators only; neither says anything about the bond |
| No prefix, no suffix | Both chain ends unmodified |
| · Cu²⁺ | Not a residue and not numbered: the copper(II) ion the tripeptide is supplied in complex with, which is why it sits outside the hyphenated string |
The counter-ion, the anion of the acid the peptide is isolated as a salt of, is likewise no part of the sequence; certificates carry a row of their own for it.
TB-500 is listed with the sequence Ac-LKKTETQ, the same exercise in the one-letter code.
| Printed | Reads as |
|---|---|
| Ac- | Prefix: the N-terminus is acetylated, and the acetyl group takes no number |
| L K K T E T Q | Leu-Lys-Lys-Thr-Glu-Thr-Gln, seven residues numbered 1 to 7 from the N-terminus |
| Nothing after Q | No suffix, so the C-terminal glutamine carries a free α-carboxyl |
| Three-letter form | Ac-Leu-Lys-Lys-Thr-Glu-Thr-Gln-OH |
TB-500 is used in the trade both for thymosin β4 and for this acetylated seven-residue fragment of it, so the sequence field, not the name, settles which molecule is described.
Where a catalog entry gives a residue count instead of a string, as Sermorelin and CJC-1295 do at 29 residues and MOTS-c at 16, the count is all that field claims; the sequence has to come from the certificate or the supplier.
Composition is not identity. Semax at MEHFPGP and Selank at TKPRPGP are both seven residues, and reversing either string gives a different molecule of the same formula and the same mass. That is why identity by ESI-MS is read alongside the written sequence, as covered in how to read a Certificate of Analysis and peptide purity by RP-HPLC and ESI-MS. Sequences are listed per compound in the catalog, and a lot’s certificate can be pulled by lot number at verify.
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