A purity assay and an identity assay answer different questions. Purity asks how much of the dominant species is present relative to everything else. Identity asks whether that dominant species is the compound named on the label. A lot can be 99% pure and 100% the wrong molecule.
Mass spectrometry answers the identity question by measuring molecular weight directly.
How electrospray ionisation produces a peptide spectrum
Electrospray ionisation, usually abbreviated ESI, sprays the sample through a charged capillary into a fine mist. Solvent evaporates from the droplets and leaves charged molecules in the gas phase, which the instrument separates by mass-to-charge ratio.
Peptides pick up protons readily, often several at once, so a single compound produces a family of peaks rather than one. A peptide of mass M appears as [M+H]+, [M+2H]2+, [M+3H]3+ and so on, each at a progressively lower mass-to-charge value. This family is called the charge-state envelope, and software deconvolutes it back to a single neutral mass.
Observed mass against theoretical mass
The theoretical mass is calculated from the sequence. Two conventions are in use and a certificate should say which:
- Monoisotopic mass uses the lightest isotope of each element. It is the convention for smaller peptides and high-resolution instruments.
- Average mass uses the natural isotopic abundance of each element. It is conventional for larger molecules and lower-resolution instruments.
Mixing the two produces an apparent discrepancy of roughly one dalton per few thousand, which is a common source of confusion when comparing a certificate against a published value.
What a mass shift usually means
Deviations between observed and theoretical mass are informative, and a handful of values recur often enough to recognise on sight.
- Plus 16 daltons indicates oxidation, most often at methionine, sometimes at tryptophan or histidine.
- Plus 1 dalton indicates deamidation, where an asparagine or glutamine side chain has converted to the corresponding acid.
- Minus the mass of one residue indicates a deletion sequence, where a coupling step failed during assembly.
- Plus 42 daltons indicates acetylation, which may be intentional at the N-terminus or an artefact.
- A doubled mass, or close to it, suggests a dimer, commonly formed through disulfide bonding between two cysteine-containing chains.
What intact mass cannot resolve
An intact-mass measurement confirms the molecular formula's total, not the order of the residues or their stereochemistry. Several real failure modes are invisible to it.
- Isomeric substitutions. Leucine and isoleucine have identical mass, so one substituted for the other produces no shift at all.
- Sequence scrambling. The same residues assembled in a different order give the same intact mass.
- Stereochemistry. A D-amino acid in place of its L counterpart is mass-identical, and racemisation during synthesis is a genuine possibility at susceptible residues.
Resolving those requires tandem mass spectrometry, written MS/MS, in which the instrument isolates the parent ion, fragments it, and reads the resulting ladder of fragment masses to reconstruct the sequence. Not every certificate includes it, and for well-characterised compounds from a consistent process it is often reserved for method validation rather than routine lot release.
What belongs on the certificate
- The theoretical mass and which convention it uses.
- The observed mass for that specific lot.
- The ionisation mode and instrument type.
- A statement of whether the result conforms to specification, and what the tolerance is.
An identity line that reads only "conforms" without an observed value is an assertion rather than a measurement. The number is the point.
Reading identity and purity together
The two assays constrain each other. A clean single peak in the chromatogram with an observed mass matching theory is a strong combined statement. A clean peak with a mass 16 daltons high says the dominant species is oxidised, which chromatography alone might have shown only as a slightly early retention time. A correct mass with a messy chromatogram says the right compound is present among a great deal else.
Neither line is sufficient alone, which is why both appear on a complete certificate, alongside the separate questions of net peptide content, endotoxin and sterility.
This sits inside the wider picture of what gets tested and why, which why peptide testing matters sets out across the whole analytical panel.