How A Peptide's Molecular Weight Is Calculated

The formation of a peptide bond between two amino acids releases one water molecule. A chain of n residues therefore has n minus 1 bonds and has lost n minus 1 water molecules relative to the free amino acids that formed it. The arithmetic used in practice inverts that: rather than subtracting, the calculation sums residue masses, which already have the water removed, and then adds a single water molecule for the terminal hydrogen and hydroxyl.

The water added back is 18.010565 Da using monoisotopic masses, or 18.01528 Da using average masses. Which of the two is used has to be decided before anything is summed, because the two conventions cannot be mixed and the gap between them grows with chain length. Monoisotopic mass uses the single most abundant isotope of each element, which is what a high-resolution mass spectrometer resolves. Average mass uses the natural isotopic abundance weighting, which is what a weighing balance and a molar calculation reflect.

For a peptide of 30 residues the two figures typically differ by roughly 1.5 to 2 Da, and for a 50-residue chain by 3 Da or more. Reported alongside a mass spectrometric result with no convention stated, a difference of that size looks like a wrong compound.

Residue masses

Leucine and isoleucine share a residue mass to five decimal places because they are constitutional isomers with identical elemental composition. No mass calculation and no mass measurement can separate them. Glutamine and lysine differ by only 0.03638 Da, which a low-resolution instrument will not resolve but arithmetic will.

Modification increments

Most synthetic sequences of interest are not plain chains with free ends, and the modifications are where calculated values most often go wrong. Each one is a fixed monoisotopic increment applied after the residue sum.

C-terminal amidation is the one that catches people, because a shift of minus 0.98 Da is close enough to a rounding error to be absorbed silently, and close enough to 1 Da to be mistaken for a proton. A calculated mass that sits about 1 Da above a measured one, on a sequence known to be amidated, is usually a forgotten amidation rather than a wrong compound.

Worked example

Take the tripeptide glycine-histidine-lysine. The monoisotopic residue sum is 57.02146 plus 137.05891 plus 128.09496, which is 322.17533 Da. Adding one water molecule at 18.010565 Da gives a free-acid monoisotopic mass of 340.18590 Da. Repeating the same arithmetic with average residue masses and average water gives 340.3789 Da. Both are correct; they answer slightly different questions, and a record that reports one while implying the other is the most common error in this arithmetic.

A mass spectrometer operating in positive mode does not report either figure directly. It reports mass-to-charge ratios of charged species, so the singly protonated form of the example above appears near 341.19318 Da, one proton at 1.00728 Da heavier. Comparing a calculated neutral mass against an observed protonated ion without accounting for the proton produces an apparent 1 Da discrepancy.

The failure mode: reading molecular weight as container contents

The calculated molecular weight describes one molecule of a defined sequence. The solid in a vial is not one molecule of a defined sequence. It is a lyophilised mixture whose mass includes the counterion carried through from purification, residual water, any bulking agent, and whatever synthesis-related species are present within the purity specification.

• Counterions add mass and are not part of the molecular weight. Trifluoroacetate contributes 113.99286 Da and acetate 59.01330 Da monoisotopic, once for every basic site they pair with.
• Residual water in a lyophilised solid is commonly in the range of 1 to 5 percent by weight, determined by Karl Fischer titration, and it is weighed along with everything else.
• A bulking agent such as mannitol, where one is present, contributes a large fraction of the total solid mass and no part of the peptide mass.
• Chromatographic purity is an area percentage from a chromatogram, not a mass fraction of the solid.

The gap between the gross mass of solid and the mass of peptide inside it is what net peptide content describes, and it is measured by amino acid analysis or by ultraviolet absorbance against an extinction coefficient, not derived from molecular weight.

Where our own position stops

Aurum publishes a molecular weight for each sequence we list, and that figure is computed from the sequence rather than measured on the lot. We publish a mass confirmation, which establishes agreement between the observed mass and the calculated one, and a reverse-phase HPLC purity figure with the wavelength stated. We do not publish net peptide content, amino acid analysis is not among the specifications Aurum publishes, and counterion or residual water from the labelled mass of solid is not among the specifications Aurum publishes. The molecular weight on a listing is therefore an identity figure and not a quantity figure, and read as the latter it answers a question it was never a record of.