Monoisotopic Mass and Average Mass: Two Numbers for One Molecule
A certificate sometimes quotes a calculated mass that does not match the number in a reference database, and both are correct. They are answers to slightly different questions, and the difference comes from how nature distributes isotopes.
Where two numbers come from
Carbon in nature is mostly carbon-12, with about 1.1% carbon-13. Hydrogen, nitrogen, oxygen and sulfur each have their own minor isotopes in their own proportions. A population of identical molecules therefore contains individuals of slightly different mass, depending on which isotopes each happens to contain.
Monoisotopic mass is the mass of the single molecule built entirely from the most abundant isotope of every element — all carbon-12, all nitrogen-14, all oxygen-16. It is one exact number.
Average mass is the abundance-weighted mean across the whole population, using standard atomic weights. It is always the larger of the two, because the minor isotopes are heavier rather than lighter.
Why the gap widens with size
The difference scales with the number of atoms. On a small peptide of a few residues it is a fraction of a dalton and easy to overlook. On a 29-residue sequence it is a dalton or two. On a 43-residue protein it can exceed three.
That matters because several of the differences worth detecting are themselves around one dalton. Deamidation shifts a mass by 0.98; an amide and a free acid differ by about one. Comparing a measured monoisotopic mass against a calculated average mass, or the reverse, produces a discrepancy of the same order as the modification being looked for. Deamidation and the 0.98 dalton shift covers that specific case.
Which instruments report which
A high-resolution instrument separates the individual isotope peaks, so the first peak in the cluster is the monoisotopic species and that is the figure reported. Modern time-of-flight and orbital-trap instruments work this way for peptides of ordinary size.
A lower-resolution instrument cannot separate the peaks. It records the envelope as a single broad signal whose centroid approximates the average mass, and that is what gets reported. MALDI and ESI covers how the two ionisation techniques differ in the spectra they produce.
For large molecules even a high-resolution instrument may not resolve the cluster, because the isotope peaks crowd together as the number of atoms grows. Average mass becomes the practical figure at that scale.
Reading a report
The useful check is that the measured and calculated figures are the same kind of number. A certificate reporting a measured mass from a high-resolution instrument against a calculated average mass will show a mismatch that has nothing to do with the material.
Charge state is the related detail on electrospray data. A reported mass-to-charge ratio is the molecular mass plus the protons, divided by the number of protons; the deconvoluted neutral mass is what compares to a calculated value. HPLC and mass spectrometry in purity verification covers how the methods divide the work.
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