What mass spectrometry can and cannot tell you

What the instrument measures

A mass spectrometer does not weigh molecules. It ionises them, sorts the ions by their mass-to-charge ratio (m/z), and counts how many arrive at each value. Every number on a mass spectrum is therefore a ratio, and the charge has to be worked out before a molecular mass can be read off it.

For peptides analysed by electrospray ionisation, that charge is frequently greater than one. A peptide of monoisotopic mass 2400 Da may appear as [M+H]⁺ near m/z 2401, as [M+2H]²⁺ near 1201, and as [M+3H]³⁺ near 801. Seeing several of these is a good sign: the charge-state series is internally consistent, and consistency across states is stronger evidence than one peak alone.

Monoisotopic versus average mass

Two different masses are routinely quoted, and comparing the wrong pair produces phantom discrepancies:

• Monoisotopic mass uses the lightest stable isotope of each element. It is what a high-resolution instrument reports for the first isotopic peak.
• Average mass weights each element by natural isotopic abundance. It is what a low-resolution instrument or a simple calculator reports.

For a peptide of a few thousand daltons the two can differ by more than a dalton — enough to look like a failed identity check when nothing is wrong. A well-written report states which convention it is using.

What a matching mass establishes

A match tells you the sample contains something whose elemental composition is consistent with the claimed molecular formula. That is genuinely useful: it rules out a large fraction of possible substitutions, truncations and wrong compounds, since almost any change to a sequence changes the mass.

Almost. The exceptions are the reason mass confirmation is not proof of identity:

• Sequence permutations. The same residues in a different order have the same mass. Only fragmentation (MS/MS) distinguishes them.
• Isobaric residues. Leucine and isoleucine are indistinguishable by mass, and hard to separate even by fragmentation.
• Stereochemistry. A D-amino acid substitution changes biological behaviour and nothing about the mass.
• Structural isomers. Cyclic versus linear forms, and different disulfide pairings, can share a nominal mass.
• Mixtures. A correct mass in the spectrum shows the compound is present, not that it dominates. Relative abundance in a mass spectrum is a poor guide to composition, because ionisation efficiency varies widely between species.

Reading the mass section of a report

Four things make a mass result checkable:

1. The expected value, with its convention. Monoisotopic or average, and derived from a stated sequence.
2. The observed value, with the adduct and charge state. “[M+H]⁺ 1234.6” rather than “mass confirmed”.
3. The instrument and ionisation mode. A high-resolution measurement supports a much tighter tolerance than a low-resolution one, so tolerance without resolution is meaningless.
4. The spectrum itself, where possible. With an m/z axis, so the reader can see the charge-state series and the isotope structure.

“Identity: conforms” on its own is an assertion by the report’s author. It may well be correct; it simply cannot be checked, which is a different property from being true.

For the wider context of how this fits alongside chromatographic purity, see peptide testing and verification.