Peptide molecular weight and the mass spectrum

Every peptide has a molecular weight set by its sequence. It is one of the most useful numbers on a listing, because it is the one a mass spectrometer can check directly. It also comes in several versions, and mixing them up is a common source of confusion.

The unit

Molecular weight for peptides is given in daltons (Da), or the equivalent g/mol. One dalton is about the mass of one hydrogen atom. Larger peptides are often given in kilodaltons (kDa): 1 kDa = 1,000 Da.

Where the number comes from

The mass of a peptide is the sum of the masses of its amino acid residues, plus one water molecule for the free ends. Each residue in a chain weighs its amino acid's mass minus one water, because a water is released when each peptide bond forms. Residue masses run from about 57 Da (glycine) to about 186 Da (tryptophan), so a rough rule is around 110 Da per residue on average.

Rough sizes:

Length	Approximate mass
5 residues	about 500 to 700 Da
20 residues	about 2,000 to 2,500 Da
40 residues	about 4,000 to 5,000 Da

Modifications change it: an amidated C-terminus is about 1 Da lighter, an acetylated N-terminus about 42 Da heavier, a disulfide bond about 2 Da lighter.

Three versions of "molecular weight"

Version	What it is	Where it is used
Monoisotopic mass	Sum using the most common isotope of each element	High-resolution mass spectrometry
Average mass	Sum using natural-abundance average atomic weights	Listings, chemical catalogs, lower-resolution MS
Salt-form weight	Average mass plus the counterions (for example TFA or acetate)	Some product listings, and analytical weighing in the lab

For a peptide of a few thousand daltons, monoisotopic and average mass differ by around one to three daltons. The salt-form weight can be hundreds of daltons higher than the free peptide, depending on how many counterions are bound.

When a listing and a COA seem to disagree on molecular weight, check which version each one uses first.

How the mass spectrum checks it

The mass spectrometer measures the peptide's ions, not the salt form. On a COA:

• The expected mass is calculated from the sequence, as monoisotopic or average.
• The observed mass comes from the spectrum, usually after deconvolution of several charge states.
• The two should agree within the instrument's tolerance.

On a high-resolution spectrum, a single charge state shows as a cluster of peaks a fraction of a unit apart: the isotope pattern. The spacing between them reveals the charge (peaks 0.5 apart mean charge 2+, 0.33 apart mean 3+), and the first peak of the cluster is the monoisotopic one.

What a molecular weight match does not settle

It confirms composition, not sequence order or purity. And because counterions and water are not part of the measured peptide mass, it says nothing about content by weight.