Average, Monoisotopic and Most Abundant Mass: Reading Peptide Mass Spectrometry Values

Why one peptide has more than one molecular weight

Elements occur as mixtures of isotopes. Carbon is 98.93 % carbon-12 and 1.07 % carbon-13, and hydrogen, nitrogen, oxygen and sulfur have their own heavier isotopes. A single molecule is made of specific isotopes, so a mass spectrometer that resolves individual ions sees a group of peaks, each one a different isotopic composition of the same chemical formula.

The molecular weight that appears in a catalogue is the average of all of them, weighted by natural abundance. The mass of the molecule made only of the lightest isotopes is the monoisotopic mass. Both numbers are correct; they answer different questions, and a COA should say which one it reports.

Monoisotopic mass: the first peak

The monoisotopic mass is the sum of the exact masses of the lightest isotope of each atom: 12.000000 for carbon, 1.007825 for hydrogen, 14.003074 for nitrogen, 15.994915 for oxygen and 31.972071 for sulfur. For BPC-157 (C62H98N16O22) it is 1418.7042. In a high-resolution instrument the first peak of the isotope pattern sits at this value, followed by peaks about 1.003 Da apart.

Monoisotopic mass is the right number to compare for peptides up to about 2.5 to 3 kDa, where the first peak is still tall enough to see.

Average mass: the catalogue molecular weight

Average mass uses the standard atomic weights, such as 12.011 for carbon and 1.008 for hydrogen, which already include the isotope mixture. For BPC-157 it is 1419.54. Average mass is the number to use for weighing and for the molecular weight on a data sheet. It is also the number that a mass spectrometer reports when it cannot resolve the isotopes of a large molecule, and the peaks merge into one broad envelope.

The most abundant mass and why it moves with size

Every carbon has a 1.07 % chance of being carbon-13. A molecule with 62 carbons has roughly a 51 % chance of containing none, so the monoisotopic peak is the tallest. A molecule with 187 carbons, such as semaglutide, has only about a 13 % chance, and the tallest peak is one to two mass units above the monoisotopic one.

This is why the exact mass listed by public databases for large peptides is often a little higher than a strict monoisotopic calculation. When two numbers differ by about 1 Da, the cause is usually which isotope peak was picked, not a different compound.

Charge states: how to calculate the ions

In electrospray ionisation, peptides pick up protons. An ion with n protons has m/z = (M + n x 1.007276) / n. For BPC-157 the [M+H]+ ion is at 1419.711, [M+2H]2+ at 710.359 and [M+3H]3+ at 473.909. For semaglutide, with a monoisotopic mass of 4111.115, the 3+ to 6+ ions fall at 1371.379, 1028.786, 823.230 and 686.193.

Larger peptides and peptides with more basic residues carry more charges, which is why a 4 kDa peptide is observed at m/z values far below 4000.

Deconvolution and adducts

Software can convert a series of multiply charged ions into one neutral mass; this is called deconvolution. The result should agree with the expected value within the stated accuracy of the instrument, which is often 0.1 Da or better for a time-of-flight or Orbitrap system and 0.5 to 1 Da for a simple quadrupole. Extra peaks that are 21.982 or 37.956 above the [M+H]+ ion are sodium and potassium adducts.

They indicate salts in the sample, not a second compound.

Using this on a certificate of analysis

Check three things. First, the COA names the expected mass and says whether it is monoisotopic or average. Second, the measured mass is given with a unit and a plausible accuracy. Third, the difference between expected and measured mass is small compared with the instrument's accuracy and is not explained by an adduct or an isotope peak.

For modified peptides, such as those with a fatty acid side chain, the expected mass must come from the full molecular formula, not from the amino acid letters. The compound pages on this site list the formula, the monoisotopic and the average mass for each compound where a public record exists.