The molecular weight of a peptide is needed for almost every bench calculation that follows: converting milligrams to moles, making a solution of known molarity, or checking a mass spectrum. It is simple arithmetic once you know what to add up, and what to leave out.
Peptide bonds release water
Each peptide bond forms by condensation. The carboxyl group of one amino acid joins the amino group of the next, and one molecule of water (H2O) is lost. A linear peptide of n amino acids therefore has n − 1 peptide bonds and has lost n − 1 waters.
That is why the mass of a peptide is not the sum of the free amino acid masses. Adding up free amino acids overestimates the mass by about 18 g/mol for every bond.
Residue masses and the one extra water
The cleaner way to do the sum is with residue masses. A residue is the amino acid minus one water, which is the unit actually present inside the chain. Glycine, for example, is C2H5NO2 as a free amino acid and C2H3NO as a residue.
Summing residues accounts for every bond, but leaves the chain one water short. The free N-terminal –H and C-terminal –OH have to be added back:
M(peptide) = Σ M(residues) + M(H2O)
The calculator works from elemental formulas rather than from a table of residue masses. It adds the C, H, N, O and S counts of every residue, adds H2O, and then multiplies by atomic masses. That gives the molecular formula and the mass from one consistent calculation.
Average or monoisotopic mass?
Most elements exist as a mixture of isotopes. Carbon is about 98.9% 12C and 1.1% 13C. There are two useful ways to handle this:
- Average molecular weight uses standard atomic weights (C 12.011, H 1.008, N 14.007, O 15.999, S 32.06), which reflect natural isotope abundance. Use it for anything you weigh: molarity, stock solutions and amount in moles.
- Monoisotopic mass uses only the most abundant isotope of each element (12C, 1H, 14N, 16O, 32S). Use it to match the first peak of an isotope pattern in high-resolution mass spectrometry.
For small peptides the two differ by a fraction of a dalton. For larger peptides the gap grows, and the monoisotopic peak becomes a smaller part of the isotope envelope.
Modifications change the formula
Synthetic peptides are often capped. Each modification changes the elemental formula in a fixed way:
| Modification | Formula change | Average Δ (g/mol) |
|---|---|---|
| N-terminal acetylation (Ac–) | +C2H2O |
+42.04 |
| C-terminal amidation (–NH2) | +N +H −O |
−0.98 |
| Each disulfide bond (Cys–Cys) | −H2 |
−2.02 |
Worked example: GHK
Residues: Gly C2H3NO + His C6H7N3O + Lys C6H12N2O = C14H22N6O3. Adding H2O gives C14H24N6O4.
Average: 14 × 12.011 + 24 × 1.008 + 6 × 14.007 + 4 × 15.999 = 340.38 g/mol.
Monoisotopic: 14 × 12 + 24 × 1.007825 + 6 × 14.003074 + 4 × 15.994915 = 340.1859 Da. The singly protonated ion [M+H]+ appears at m/z 341.193.
Common mistakes
- Adding free amino acid masses. This overestimates by 18.02 g/mol per peptide bond.
- Forgetting the terminal groups. An amidated peptide is about 1 g/mol lighter than its free acid, and an acetylated one is 42 g/mol heavier.
- Using the salt form as if it were the peptide. Lyophilised peptides are often supplied as trifluoroacetate or acetate salts and contain some water. The certificate may quote a molecular weight that includes counter-ions, or a net peptide content below 100%. Use the right figure for the job (see preparing a stock solution).
- Weighing with the monoisotopic mass. Balances weigh natural isotope mixtures, so use the average mass.
References
- CIAAW: Standard atomic weights
- NIST: Atomic weights and isotopic compositions with relative atomic masses
- IUPAC-IUB JCBN: Nomenclature and symbolism for amino acids and peptides
- Murray KK et al. (2013). Definitions of terms relating to mass spectrometry (IUPAC Recommendations 2013). Pure Appl Chem 85:1515–1609
- ExPASy ProtParam documentation