Peptide Sequence Notation Explained

One-Letter and Three-Letter Amino Acid Codes

The twenty standard amino acids that make up ribosomally encoded proteins and most synthetic research peptides each have a standardized one-letter code and a three-letter code. The three-letter code is generally easier to read without ambiguity; the one-letter code is more compact and is what appears in most sequence databases and synthesis order sheets.

A sequence written as three-letter codes separated by hyphens, for example Tyr-Gly-Gly-Phe-Leu as an illustrative fragment, and the equivalent one-letter string, YGGFL, describe the same order of residues. The choice between formats is a matter of convention in a given document, not a difference in meaning.

N-Terminus to C-Terminus: Reading Direction Matters

Peptide sequences are written, by universal convention, from the N-terminus (the end with a free amino group) to the C-terminus (the end with a free carboxyl group), left to right. This is not an arbitrary typesetting choice. Reversing the order of the same amino acids produces a genuinely different molecule, because the direction of the peptide bonds and the resulting side-chain spacing along the backbone changes with it.

A sequence such as GHK, written N-terminus to C-terminus as an illustrative example of a short tripeptide, is not the same molecule as the same three letters read in reverse order. When comparing a stated sequence against a reference sequence, direction has to match, not just composition. This is also why sequence documentation should always make clear which end is which when there is any risk of ambiguity, rather than assuming the reader will infer it.

N-Terminal Acetylation

Acetylation at the N-terminus replaces the free amino group at that end with an acetyl group, which removes the positive charge normally present there at physiological pH and changes how the N-terminal end of the peptide interacts with its surroundings. In sequence notation this is typically indicated with an "Ac-" prefix before the first residue, for example Ac-Ser-Tyr as an illustrative fragment. Acetylation adds a small, fixed mass increase to the molecule, and a mass spectrometry result for an acetylated peptide should show a molecular weight consistent with the unmodified sequence plus that increment, not the unmodified sequence's plain mass.

C-Terminal Amidation

Amidation at the C-terminus replaces the free carboxyl group at that end with an amide group, removing the negative charge normally present there and changing the overall charge profile of the molecule. This is commonly indicated with a "-NH2" suffix after the final residue, for example Gly-NH2 as an illustrative notation. Like acetylation, amidation changes the molecular weight by a small, fixed, well-known amount relative to the unmodified free-acid form of the same sequence, and that shift should be visible and consistent in a mass spectrometry identity check.

Cyclization and Disulfide Bridges

Some peptides are cyclic rather than linear, meaning the backbone forms a closed ring rather than running from a free N-terminus to a free C-terminus. Cyclization can occur head-to-tail, connecting the N-terminus directly to the C-terminus, or through side-chain linkages between two residues elsewhere in the sequence.

A disulfide bridge is a specific type of side-chain linkage formed between the sulfur atoms of two cysteine residues, either within a single chain to create a loop, or between two separate chains to link them together. Disulfide bridge formation removes two hydrogen atoms relative to the reduced, unlinked form of the same sequence, which produces a small, specific, and predictable mass decrease that should be visible on mass spectrometry as confirmation the bridge actually formed rather than the cysteines remaining in their free, reduced state. Documentation for a cyclic or disulfide-bridged peptide should state which residues are connected, since the same amino acid composition with a bridge in a different position is again a different molecule.

D-Amino Acids and Non-Standard Residues

The amino acids listed in the standard reference table are, by default, the L-isomer form, which is the form used in naturally occurring proteins. A D-amino acid is the mirror image isomer, and substituting one into a sequence changes the molecule's three-dimensional shape and its stability against certain enzymatic breakdown, without changing its molecular weight relative to the L-form at that position. Because D-substitution is invisible to mass alone, it has to be called out explicitly in sequence notation, conventionally with a lowercase letter or a "D-" prefix on that specific residue, for example D-Ala at a given position.

Non-standard residues, amino acids outside the twenty-residue standard set, such as ornithine, norleucine, or various synthetic and unnatural side chains used in some research peptides, do not have a place in the standard one-letter code and are instead spelled out by name or given a defined abbreviation specific to that sequence's documentation. Any sequence containing a non-standard residue should define that residue explicitly rather than leaving a reader to guess what an unfamiliar abbreviation means.

PEGylation and Fatty-Acid Acylation

PEGylation attaches one or more polyethylene glycol (PEG) chains to a peptide, usually at a specific side chain or terminus. PEG chains come in a range of defined lengths, and the length used has to be stated as part of the identity of a PEGylated peptide, because it adds a large and variable mass increase that dwarfs the mass of the underlying peptide chain in many cases. A PEGylated peptide's mass spectrometry result will typically show either a distinct shifted mass, or in some cases a spread of peaks corresponding to a distribution of PEG chain lengths around a nominal average, depending on how uniform the PEG reagent used was.

Fatty-acid acylation attaches a fatty-acid chain, usually through a linker, to a specific side chain, most often a lysine residue. This is a distinct, well-known modification class in peptide chemistry, and like PEGylation, it adds a specific, calculable mass increment on top of the base peptide. The identity of the fatty-acid chain and its attachment point are both part of what makes the resulting molecule a specific, defined compound rather than a generic modified peptide.

Why Sequence Plus Modifications Is the Real Identity

A trade name or product name is a label a supplier or the broader research community has attached to a specific combination of sequence and modification set. The name itself carries no independently verifiable chemical information. The sequence, written correctly with terminus direction and every modification stated, is the actual chemical description of the molecule, and it is the only part of the documentation that can be independently checked against an analytical result.

Two products marketed under similar or identical names from different suppliers are not guaranteed to be the same molecule unless their stated sequences and modification sets match exactly. This is precisely why a sequence and modification list, not a trade name, belongs on a rigorous certificate of analysis, and why a buyer comparing two suppliers should compare that underlying description rather than assuming a shared name means a shared structure.

Checking a Stated Sequence Against Molecular Weight and Formula

A sequence, once every modification is accounted for, has a calculable theoretical molecular weight and a calculable molecular formula, both derivable from the known mass and formula contribution of each residue and each modification. A buyer reviewing a certificate of analysis can check, at a basic level, whether the stated molecular weight is at least plausible for the stated sequence and modification set, using published residue mass tables as a reference.

More importantly, the mass spectrometry result on the certificate should match the molecular weight implied by the stated sequence and its modifications, within the normal precision of the instrument used. A mismatch between the stated sequence and the observed mass is one of the clearest, most objective signs that something is wrong, either an error in the stated sequence, an unstated modification, or a material that is not what the label claims. This cross-check does not require independent lab access. It only requires reading the sequence carefully enough to know what modifications are claimed, and comparing the resulting expected mass against what the mass spectrometry section of the same certificate actually reports.