Peptide Sequence: Notation, Methods and Identity
TL;DR · The short version
A peptide sequence gives residue order, conventionally from N-terminus to C-terminus. Keep stereochemistry and modifications with that record. A listed sequence describes the intended structure; an analytical assignment depends on what the method actually examined.
GHK and KHG contain the same three letters but describe different peptide sequences. A matching molecular weight won’t distinguish that rearrangement on its own.
Read the order first, then the end groups and modifications. Finally, check the analytical evidence. Small annotations can be part of the chemical identity even when they sit outside the familiar letters.
Start with the two ends
TL;DR: Read the chain from N to C unless the source explicitly defines another notation.
The N-terminus is the amino end and the C-terminus is the carboxyl end. Sequences are conventionally written N to C. IUPAC peptide nomenclature distinguishes the residue chain and its terminal groups. For a modified or cyclic peptide, the source should define how those features are represented.
Gly-His-Lys and Lys-His-Gly contain the same three residues in different positions. Their composition-based formulas can match despite that difference. This is why molecular weight can’t answer every sequence question.
Read a short peptide sequence step by step
TL;DR: GHK means Gly-His-Lys: three residues in that order, with two peptide bonds in the ordinary linear chain.
Read GHK one position at a time: glycine first, histidine second, lysine third. Three residues make it a tripeptide. In the ordinary unmodified linear chain, two peptide bonds connect them.
Residue order is primary structure, not a complete picture of the chain’s conformation in solution. For a fragment, also retain the parent identity and residue numbering so its position remains clear.
Keep modifications attached to the sequence
TL;DR: Retain stereochemistry, terminal chemistry, ring connections and attached groups.
| Feature | Why it matters when comparing records |
|---|---|
| D/L configuration | The stereochemical form is part of residue identity; do not remove a D- prefix. |
| Terminal modification | Acetylated or amidated ends are different from the corresponding free termini. |
| Cyclization or disulfide bond | Connectivity may not be fully represented by the linear string. |
| Attached group | A linker, lipid or other conjugate belongs in the complete identity. |
| Counterion or associated water | These affect the material description and mass basis without becoming chain residues. |
How is a peptide sequence determined?
TL;DR: Edman sequencing and MS/MS obtain order-related evidence. An intact-mass measurement asks a different question.
Sequencing obtains evidence about order. Calculating mass from a proposed sequence or measuring amino-acid composition answers a different question.
Edman degradation reads from the N-terminus
Edman chemistry identifies successive residues by removing one at a time from the N-terminal end. It requires an accessible, unmodified terminal amino group; a blocked end can prevent the ordinary reaction. The Wistar Institute authors’ N-terminal sequencing protocol explains this cycle and its practical limits. A partial read establishes only the segment successfully examined, not automatically the entire chain.
Tandem mass spectrometry examines fragments
In MS/MS, a selected peptide ion is fragmented and the resulting ions are measured. Those fragments can provide evidence about the sequence. The University of Washington’s targeted proteomics guide describes the distinction between a peptide precursor ion and its sequence-related fragment ions. Measuring the intact precursor alone is a different experiment.
| Evidence in a record | What to check |
|---|---|
| Intact molecular mass | Whether it agrees with the proposed complete chemical form; agreement alone does not establish residue order. |
| N-terminal sequence read | Which positions were read and whether a blocked terminus or incomplete read limits the result. |
| MS/MS fragment assignments | Which parts of the proposed sequence the assigned fragments support, and which distinctions remain unresolved. |
Separate a specified sequence from an experimentally supported sequence
TL;DR: A specified structure and an experimentally supported assignment are different entries in the record.
Keep the catalogue specification separate from the submitted sample’s result. Was the result intact mass, chromatographic purity or fragment-ion evidence? Its method determines which part of the identity claim it supports.
A fragment-based assignment does not automatically resolve every residue isomer, stereochemical distinction or structural ambiguity. The peptide reference-standard characterization study describes the value and limits of complementary methods. Interpret the stated method rather than upgrading a simple mass match to “complete sequence verified.”
Compare like with like
TL;DR: Check fragments, termini, form and transcription before declaring a mismatch.
For a mismatch, check whether one record describes a fragment, terminal modification or salt form. Then check direction and transcription. Similar names may conceal different structures; different names may sometimes refer to the same specified entity.
When literature uses a name inconsistently, leave that issue open until the supplied material is documented. Choosing the sequence with the closest mass doesn’t establish that it is the right one.
What to record in a research material entry
TL;DR: Keep the notation, source and lot together, with unresolved details clearly marked.
Save the notation, modifications, form, source and lot together. Flag missing information. The next reviewer should be able to follow the evidence without reconstructing an assumption you left unstated.
For a code reference beside the sequence, use our amino acid abbreviations guide. To understand why a matching mass is not a complete sequence assignment, see peptide molecular weight.