Average vs Monoisotopic Mass

Monoisotopic mass is the mass of a molecule made only of the most abundant isotope of each element; average mass uses natural-abundance atomic weights and is what a molecular weight on a label means; nominal mass rounds to integers. The gap comes mostly from carbon-13, about 1.1 percent of carbon and 1.003 Da heavier, and for peptides runs near 0.06 percent. Semaglutide, C187H291N45O59, is about 4113.6 average and 4111.1 monoisotopic, a gap of 2.5 Da.

With 187 carbons only about one molecule in ten is entirely light, so the monoisotopic peak is minor and the tallest peak sits two mass units higher. A high-resolution instrument resolves the peaks at their 1.003 Da spacing and reports the monoisotopic mass. A low-resolution quadrupole sees one unresolved hump centred near the average mass.

The first question about any mass-spectrometry result is which mass it is compared with. Found 4113.5 against calculated 4113.6 is a low-resolution centroid against the average mass; found 4111.12 is a high-resolution monoisotopic match. Mixing the two manufactures a 2.5 Da discrepancy where none exists, or hides a real one of the same size. The spectrometer also never sees a counterion, so a salt-form molecular weight is the wrong reference by 60 Da per acetate.

A C-terminal amide is 0.984 Da lighter than the acid, and deamidation of asparagine adds the same 0.984 Da. Since isotope peaks sit 1.003 Da apart, the acid's monoisotopic peak lands almost on the amide's first heavy peak, and at 4 kDa the unresolved envelopes overlap almost perfectly. A low-resolution identity check therefore confirms the sequence mass to about a dalton and nothing about the terminus or deamidation; only a resolved isotope pattern or sub-0.1 Da accuracy can.