Abstract
Mass spectrometry provides the identity confirmation that chromatographic purity cannot supply. This article covers ionisation, charge states, mass accuracy, and the interpretation of common mass differences.
Chromatography establishes composition; mass spectrometry establishes identity. By measuring the mass-to-charge ratio of ionised molecules, it determines whether the dominant species in a sample corresponds to the intended sequence, and often what the minor species are.
Ionisation and charge states
Peptides are typically ionised by electrospray, in which a solution is sprayed through a charged capillary and solvent evaporates to leave gas-phase ions. Because peptides carry multiple ionisable groups, a single compound usually appears at several charge states. Each observed signal must be converted to a neutral monoisotopic or average mass before comparison with the theoretical value.
- Soft ionisation preserves the intact molecule rather than fragmenting it
- Multiple charge states are expected and mutually confirmatory
- Isotope spacing indicates the charge state of a given signal
- Adducts with sodium or potassium appear at predictable offsets
Mass accuracy and what it supports
The strength of an identity confirmation depends on the accuracy of the instrument. Agreement within a few parts per million on a high-resolution instrument strongly supports the proposed composition. Agreement within a fraction of a dalton on a lower-resolution instrument is consistent with the proposed sequence but does not exclude every isobaric alternative.
Mass spectrometry confirms mass; sequence assignment follows from mass together with the known synthetic route or from fragmentation data.
Interpreting mass differences
Deviations from the expected mass are often diagnostic. An increase of sixteen daltons commonly indicates oxidation; a decrease of one dalton with an increase of one in another position suggests deamidation; losses corresponding to a single residue mass point to deletion sequences from incomplete coupling. These patterns allow related substances observed chromatographically to be assigned probable structures.
Combining the techniques
Liquid chromatography coupled to mass spectrometry provides both dimensions simultaneously: components are separated in time and identified by mass as they elute. This combination is the standard basis for peptide characterisation reports, and it is why identity and purity determinations normally appear on the same document.
