Average Mass And Monoisotopic Mass Are Different Numbers

Carbon is the reason. Carbon-12 has a mass of exactly 12.000000 u by definition and a natural abundance of about 98.93 atom percent. Carbon-13 has a mass of 13.00335 u and an abundance of about 1.07 atom percent, figures published by the IUPAC Commission on Isotopic Abundances and Atomic Weights. Weight those two together and the standard atomic weight of carbon is 12.011 u.

A molecule containing 180 carbon atoms therefore has a monoisotopic mass calculated with 12.000000 for every carbon, and an average mass calculated with 12.011 for every carbon. The difference from carbon alone is 180 multiplied by 0.011, or about 2.0 u. Nitrogen-15, oxygen-18, sulfur-34 and deuterium add smaller contributions in the same direction.

Neither number is an approximation of the other. They answer different questions. Monoisotopic mass asks what one specific isotopologue weighs. Average mass asks what a large population of the molecule weighs on average.

The gap grows with molecular size

Per residue the difference is a few hundredths to a tenth of a dalton. Across a chain it accumulates. As a working rule the average mass of a peptide exceeds its monoisotopic mass by roughly 0.05% to 0.06% of the molecular weight, which means about 0.5 to 0.6 Da at 1,000 Da and about 2.0 to 2.4 Da at 4,000 Da.

Which one an instrument reports

The measurement decides which number comes out. An instrument with enough resolving power to separate the carbon-13 peaks reports individual isotopologues, and the lightest resolved peak is the monoisotopic one. Resolving adjacent isotope peaks separated by about 1 Da requires resolving power on the order of 10,000 at full width half maximum for a small peptide, and much more as mass rises.

An instrument that cannot resolve them reports a single broad peak whose centroid approximates the average mass. Linear-mode MALDI time-of-flight above a few thousand daltons behaves this way. Reflectron-mode time-of-flight, Orbitrap and Fourier transform ion cyclotron resonance instruments resolve the envelope and give monoisotopic figures.

The failure mode: comparing across conventions

The characteristic error is an observed monoisotopic mass compared against a catalogue average mass, or the reverse. At 4,000 Da that mismatch is about 2 Da in a predictable direction: the observed monoisotopic figure comes in about 2 Da below the quoted average figure, and the material looks 2 Da light.

Two Da is not a harmless offset. It is exactly the mass change from forming one disulfide bond, which is a loss of 2.016 Da. So a correctly folded, single-bridge peptide measured monoisotopically against an average-mass reference for the reduced chain lands close to the value expected for the reduced chain compared monoisotopically, and the two errors partly cancel. The arithmetic agrees and the conclusion is wrong.

• A 2 Da discrepancy at 4,000 Da is the expected size of a convention mismatch, not evidence of a modification.
• A +16 Da or +32 Da discrepancy is not explained by convention and points at oxidation.
• A +42 Da discrepancy is not explained by convention and points at acetylation.
• Convention mismatch scales with molecular weight; chemical modifications do not.

The way out is to state the convention alongside every figure, and to compare like with like. A calculated mass should carry the word monoisotopic or average, and an observed mass should carry the instrument mode that produced it. Where neither is stated, the discrepancy cannot be interpreted at all.

What neither number contains

Both conventions describe the neutral molecule of the stated formula. Neither accounts for what else is in the vial. A peptide isolated as a trifluoroacetate or acetate salt carries counter-ions whose mass is not part of either figure, and adsorbed water is not part of either figure. So a mass that matches perfectly says nothing about how much of the weighed powder is peptide.

Modification also moves both numbers in step. Attaching a polyethylene glycol chain raises average and monoisotopic mass alike, and because a polymer is a distribution rather than a single species the observed envelope is broad for reasons that have nothing to do with isotopes.

Where our own record stops

Where Aurum publishes a molecular weight it is the average mass, calculated from the sequence and standard atomic weights for the free peptide. That figure is not what a high-resolution spectrum reports, and a monoisotopic measurement should be expected to sit below it by roughly 0.05% of the mass. Monoisotopic masses are not among the specifications Aurum publishes, nor are accurate-mass figures with a stated error in parts per million, nor elemental composition confirmations or salt-corrected molecular weights.

Naming the convention is the whole of the value here. A molecular weight without a convention attached is not a specification, and we would rather say which one ours is than leave the difference to be discovered against an instrument reading.

Common questions

Is monoisotopic mass more precise than average mass?

It is defined more tightly, because it depends on isotope masses that are known to many decimal places rather than on natural abundances that vary slightly by source. It is not more correct. For a bulk powder on a balance, average mass is the relevant figure.

Why is carbon listed as 12.011 in one place and 12.000 in another?

Because 12.000000 u is the mass of the carbon-12 nuclide and 12.011 u is the standard atomic weight of natural carbon, which contains about 1.07 atom percent carbon-13. Both are published by IUPAC and neither is a rounding of the other.

Does the difference matter below 1,000 Da?

It is about 0.5 Da at 1,000 Da, which is below one nominal mass unit and therefore invisible if masses are compared as integers. It becomes visible as soon as figures are quoted to one decimal place.

What is the monoisotopic mass of a protonated ion?

The neutral monoisotopic mass plus 1.00728 Da per proton, which is the proton mass rather than the hydrogen atom mass. Using 1.00783, the hydrogen atom, introduces an electron mass of error per charge and is a recognisable small discrepancy in reported figures.

Which convention should a certificate use?

Whichever one the measurement produced, stated explicitly. A certificate that reports an observed mass and a calculated mass without saying which convention each uses cannot be checked, and an unfalsifiable figure is not a specification.