Peptide Modifications and Salt Forms: A Plain-English Guide
For laboratory research use only. Not for human consumption.
A peptide’s sequence is only part of its description. The same chain of amino acids can carry a modified end, an internal bridge or a ring, and it can be supplied as the free peptide or as a salt. Each of these changes the molecular formula and the molecular weight. This guide explains, in general chemistry terms, how the common modifications and salt forms are written and what each one does to the numbers. It covers chemistry and terminology only.
A quick recap: residues and ends
A peptide is a chain of amino acid residues joined by peptide (amide) bonds, and by convention it is written from the N-terminus (free amino group) on the left to the C-terminus (free carboxyl group) on the right. Most of the modifications below change one of those two ends, or link parts of the chain together.
N-terminal acetylation: “Ac-“
In N-terminal acetylation, an acetyl group (CH3CO–) is attached to the amino group at the start of the chain. In the three-letter system it is written as the prefix “Ac-“, for example Ac-Gly.
Chemically, an acetyl group replaces one hydrogen atom on the nitrogen, so the formula increases by C2H2O, about 42.04 g/mol.
Worked example. Glycine is C2H5NO2 (75.07 g/mol). N-acetylglycine is C4H7NO3 (117.10 g/mol). The difference, 42.03 g/mol, matches the added C2H2O within rounding.
C-terminal amidation: “-NH2”
In C-terminal amidation, the –OH of the final carboxyl group is replaced by –NH2, turning the acid into an amide. It is written as the suffix “-NH2”, for example Gly-NH2 (glycinamide). IUPAC notes that this suffix should not be confused with an amino group on a side chain, which is written differently.
Swapping –OH for –NH2 changes the formula by one more hydrogen and nitrogen and one less oxygen, so the mass falls by just under 1 g/mol.
Worked example. Glycine (C2H5NO2, 75.07 g/mol) becomes glycinamide (C2H6N2O, 74.08 g/mol): a difference of 0.99 g/mol, which matches the −0.98 change within rounding.
Because this change is so small, the molecular weights of an amidated peptide and the matching free acid sit very close together, even though they are different compounds.
Disulfide bridges
Two cysteine residues can be joined through their sulfur atoms. One hydrogen atom is removed from each –SH group, and the two sulfur atoms form an S–S bond. Two linked cysteines are known as cystine.
So each disulfide bridge lowers the formula by two hydrogen atoms, about 2.02 g/mol.
Worked example. Two molecules of cysteine (C3H7NO2S, 121.16 g/mol each) add up to 242.32 g/mol. Cystine is C6H12N2O4S2 at 240.3 g/mol: two hydrogens lighter.
Cyclic peptides: “cyclo(…)”
A peptide closed into a ring by a peptide bond between its two ends is written with the sequence in brackets after “cyclo”, for example cyclo(-Gly-Gly-). Rings can also be closed through other links, such as a disulfide bridge.
Closing a ring head to tail forms one extra peptide bond, so one more molecule of water (18.015 g/mol) is lost.
Worked example. Glycylglycine (Gly-Gly) is C4H8N2O3, 132.12 g/mol. Its head-to-tail cyclic form, cyclo(Gly-Gly), also known as 2,5-diketopiperazine, is C4H6N2O2 at 114.10 g/mol, one water lighter.
D-amino acids: same formula, different molecule
Apart from glycine, the standard amino acids exist as two mirror-image forms, L and D. A D-residue in a synthetic peptide is shown with a “D-” prefix, for example Phe-D-Ala-Leu.
Swapping an L-residue for its D form does not change the formula or the molecular weight. L-alanine and D-alanine are both C3H7NO2 at 89.09 g/mol. This is another case where molecular weight alone cannot tell two different peptides apart.
Salt forms and counter-ions
Some amino acid side chains are basic (those of lysine, arginine and histidine), as is the free amino group at the N-terminus. Basic groups can carry a positive charge, so peptides are often isolated as salts, paired with negatively charged counter-ions.
Where common counter-ions come from
Trifluoroacetic acid (TFA) is widely used in solid-phase peptide synthesis, both to release the finished peptide from its solid support and during purification. As a result, peptides with basic groups are often first obtained as TFA salts. They may later be exchanged to another counter-ion, commonly acetate or chloride.
Salt names follow the parent compound, for example “lysine monohydrochloride” or, for a peptide with two basic groups, a “dihydrochloride”.
Why a salt form changes the stated mass
A counter-ion is not part of the peptide sequence, but it is part of the material, so it adds to the formula and the molecular weight. The salt also has its own database entry: PubChem lists glycylglycine and glycylglycine hydrochloride as separate compounds with separate formulas and masses.
Worked example (arithmetic only).
| Form | Formula | Molecular weight (g/mol) |
|---|---|---|
| Glycylglycine (free peptide) | C4H8N2O3 | 132.12 |
| Glycylglycine hydrochloride | C4H9ClN2O3 | 168.58 (132.12 + 36.46 for HCl) |
| Glycylglycine with one TFA (calculated) | C4H8N2O3 · C2HF3O2 | about 246.14 (132.12 + 114.02 for TFA) |
A peptide with several basic groups can carry several counter-ions, and the gap grows with each one.
As well as the peptide and its counter-ions, a freeze-dried powder can also contain residual water. This is why two descriptions of “the same” peptide can quote different formulas and molecular weights depending on the salt form they describe.
Quick reference
| Feature | How it is written | Change to the formula | Approx. change in mass |
|---|---|---|---|
| N-terminal acetylation | Ac- at the start | + C2H2O | + 42.04 g/mol |
| C-terminal amidation | -NH2 at the end | + NH, − O | − 0.98 g/mol |
| Disulfide bridge | S–S link between two Cys | − 2 H | − 2.02 g/mol |
| Head-to-tail ring | cyclo(…) | − H2O | − 18.02 g/mol |
| D-residue | D- prefix | none | none |
| Salt form | e.g. acetate, hydrochloride, trifluoroacetate | + counter-ion(s) | depends on the counter-ion and how many |
Summary
Modifications such as acetylation, amidation, disulfide bridges and ring closure each change a peptide’s formula and molecular weight in a predictable way, while a D-residue changes the structure without changing the mass. Salt forms add counter-ions to the material, so a salt always weighs more than the free peptide it contains. When two descriptions of a peptide disagree on formula or mass, the modifications and the salt form are the first places to look.
For how lyophilised material is kept in the lab, see our storage and stability guide.
Related reading
- Peptide Sequence, Structure and Molecular Weight: A Plain-English Guide
- Storage and Stability of Lyophilised Peptides
- Lyophilised Powder vs Solution: What’s the Difference?
- Research materials
For laboratory research use only. Not for human consumption.