Mass Accuracy, Parts Per Million, and What a Mass Match Establishes
A mass spectrum returns a number, and a certificate compares it to a calculated one. How close they need to be before the comparison means something depends on what the instrument can do, and that is expressed in parts per million rather than in daltons.
Why the unit is relative
An instrument’s error is roughly proportional to the mass being measured, so quoting it as a fixed number of daltons is misleading across a range. Parts per million expresses it as a fraction: an error of 5 ppm is 5 millionths of the measured value.
The consequence is that the same instrument specification means a different absolute tolerance for different molecules. At 5 ppm, a 1,000 dalton peptide is measured to within 0.005 daltons; a 5,000 dalton peptide to within 0.025. Both are well inside the one-dalton differences that matter for amidation or deamidation — on an instrument capable of that specification.
A lower-resolution instrument quoting accuracy in the hundreds of ppm cannot resolve those differences at all. At 200 ppm a 1,000 dalton peptide is measured to within 0.2 daltons, which is adequate to confirm the compound is approximately the right size and inadequate to confirm which terminal form it is.
Calibration is what makes the number meaningful
Mass accuracy is not an intrinsic property of an instrument; it is a property of an instrument that has been calibrated recently against standards of known mass. Calibration drifts with temperature and with use.
Internal calibration — including a reference compound in the same acquisition as the sample — is more reliable than external calibration performed earlier in the day, because it corrects for drift at the moment of measurement. Which was used is rarely stated on a customer-facing report. Reference standards and traceability covers the wider principle.
What a mass match establishes
That the measured mass is consistent with the molecular formula calculated for the claimed sequence. That is a meaningful constraint and it is not an identification.
Several things share a mass. Any rearrangement that conserves the formula — a sequence isomer with the same residues in a different order, an isoaspartate rearrangement, a disulfide isomer with different connectivity, a peptide containing a D-residue — is indistinguishable by mass alone. Racemisation and chiral purity and disulfide bonds and scrambling cover two of those cases.
Distinguishing them requires fragmentation — breaking the molecule and measuring the pieces, which reports sequence rather than composition — or an orthogonal separation. Neither is part of a routine identity package.
Reading the figure
Where a report gives measured and calculated masses, the difference between them expressed in ppm is the informative quantity, and a report that states it is telling you something a report quoting two bare numbers is not. Monoisotopic and average mass covers the prerequisite: that both figures are the same kind of number before any comparison is drawn.
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