A certificate of analysis arrives, the observed mass is two daltons off the expected one, and the question is whether that matters. Often it does not — the two numbers were calculated on different bases. Sometimes it matters a great deal. Telling the two cases apart takes three things: knowing which mass you are comparing against, knowing what the instrument actually measured, and recognising the handful of mass differences that recur. This page covers all three.
Every peptide has two masses that are both correct. The monoisotopic mass sums the lightest isotope of each element — 12C, 1H, 14N, 16O. The average mass uses the natural abundance-weighted atomic weights. They are not interchangeable, and the gap grows with the peptide.
| Peptide | Residues | Monoisotopic | Average | Difference |
|---|---|---|---|---|
| Angiotensin II | 8 | 1045.53 | 1046.18 | 0.65 |
| Substance P (free acid) | 11 | 1347.71 | 1348.62 | 0.91 |
| Glucagon | 29 | 3480.62 | 3482.75 | 2.13 |
| A 50-mer | 50 | 5931.75 | 5935.68 | 3.93 |
The divergence runs at roughly 0.65 Da per 1000 Da of peptide, or about one dalton for every 1500. For a 29-residue peptide that is a 2.1 Da discrepancy produced by nothing more than an arithmetic convention. High-resolution instruments can resolve the isotope pattern and so report a monoisotopic mass; lower-resolution instruments and large peptides are better compared on the average basis. Reports may quote the monoisotopic, most-abundant or deconvoluted average mass, so the basis is worth checking before comparing. If a reported mass is two units high on a peptide of a few thousand daltons, check the basis before investigating the chemistry.
Electrospray produces ions carrying several protons, and the detector records mass divided by charge. A peptide therefore appears not as one peak but as a ladder of them, one per charge state. For a neutral monoisotopic mass M carrying z protons:
| m/z | = (M + z × 1.00728) / z |
| M | = z × (m/z) − z × 1.00728 |
| charge from isotope spacing | z = 1.00336 / Δ(m/z) |
The constant 1.00728 is the proton mass; 1.00336 is the 13C–12C difference. The third line is the useful one at the bench: if adjacent isotope peaks within a cluster are 0.5 units apart, the ion is doubly charged; a third of a unit apart, triply charged. Each charge state in the ladder should deconvolute to the same neutral mass, and that consistency is itself the check — a single peak that refuses to agree with the rest of the envelope is usually an impurity or an adduct rather than the product.
One consequence catches people out. Because the isotope envelope shifts toward heavier peaks as a molecule grows, the monoisotopic peak is the tallest only for small peptides. By a few thousand daltons it is no longer the most intense peak in its own cluster, and for large peptides it may not be visible at all — which is precisely why average mass becomes the sensible basis there.
Ionisation artefacts. Sodium replacing a proton adds 21.98 Da, potassium 37.96, and residual trifluoroacetate from reversed-phase purification associates at 113.99. These say nothing about the peptide; they say the sample needs desalting. The diagnostic test is simple: desalt and reinject. A shift that survives desalting is unlikely to be a simple adduct, though some adducts are persistent enough to survive a desalting step, so a single round is suggestive rather than conclusive.
Real chemistry. A loss of 18.01 is water — which, on a sequence containing an Asp-Xaa motif, should immediately raise the question of aspartimide formation. A loss of 17.03 from an N-terminal glutamine is pyroglutamate. Oxidation adds 15.99 and points at methionine or tryptophan. Acetylation adds 42.01.
Protecting groups left behind. A gain of 56.06 is a tert-butyl group, 100.05 a Boc, 242.11 a trityl, 252.08 a Pbf. These mean the cleavage was incomplete, and they are strong evidence about which residue is involved: Pbf belongs to arginine, trityl to cysteine, histidine or asparagine. For a fuller list, see our modification mass table; for why the cleavage cocktail matters here, see cleavage cocktails and scavengers.
One case that requires particular care. Deamidation of asparagine or glutamine adds 0.984 Da. The first 13C isotope peak of the unmodified peptide sits 1.003 Da above the monoisotopic peak. The two are separated by only 19.34 mDa, and the 13C peak of the amidated form is routinely misassigned as the monoisotopic peak of the deamidated one — a well-documented source of false positives in proteomics that requires high resolving power to resolve1. A deamidation claim resting on low-resolution intact mass alone should be treated with caution, because the 0.984 dalton shift sits very close to the spacing of the first carbon-13 isotope peak. It can still be supported, but by complementary evidence — higher resolving power, chromatographic behaviour, or peptide mapping — rather than by the intact mass on its own.
Mass is a weak constraint, and the things it cannot distinguish are exactly the things that most often go wrong in a synthesis.
This is the reason a mass spectrum is always read alongside an HPLC trace rather than instead of one. The chromatogram answers "how much of the detected material is one thing?" — bearing in mind that peak area reflects detector response rather than mass directly, and that coelution hides components; the mass spectrum answers "and is that thing the right formula?". Neither question is sufficient on its own.
Every LifeTein peptide ships with its HPLC chromatogram and mass spectrum, so the arithmetic above can be done rather than taken on trust. If a result does not look right, send it to us and we will work through it with you.