Peptides are difficult to get into the gas phase: they are large, polar, and not volatile, so they do not evaporate. Two ionisation techniques solve this and dominate peptide work.

Electrospray ionisation passes a solution of the sample through a fine needle held at high voltage, producing a spray of charged droplets. As solvent evaporates, the droplets shrink until ions are released into the gas phase. Electrospray tends to add multiple protons to a peptide, producing a series of ions at different charge states rather than a single signal.

Matrix-assisted laser desorption ionisation embeds the sample in a solid matrix that absorbs laser energy. A laser pulse vaporises a small region, carrying sample molecules into the gas phase with it. This approach typically produces singly charged ions, which makes the resulting spectrum simpler to read.

Because electrospray produces multiply charged ions, a single peptide appears at several m/z values at once. A peptide of mass M carrying two protons appears near M divided by two; carrying three, near M divided by three. Each of these is a different reading of the same molecule.

The molecular mass is recovered by deconvolution, which works backward from the observed series to the single mass consistent with all of them. The presence of a coherent charge-state series is itself informative, because the spacing between the peaks constrains what mass could have produced them.

Two conventions exist for the expected value. Monoisotopic mass is calculated using the most abundant isotope of each element. Average mass accounts for the natural isotopic distribution. The two differ measurably for a peptide, so a document reporting an observed mass should make clear which convention the expected value follows.

A peptide's mass is determined by its amino acid composition. When the observed mass matches the value calculated from the intended sequence within the instrument's tolerance, that is meaningful evidence that the material is the intended compound, and it is the basis for the identity statement on an analytical document.

The support is strong but not absolute. Mass reflects composition alone, so sequences containing the same residues arranged differently share a mass. Certain residue substitutions also happen to be nearly mass-neutral. Resolving these cases requires tandem mass spectrometry: an ion is fragmented and the fragments are measured, giving information about the sequence as well as the total mass.

For routine identity confirmation, the standard combination is a matching intact mass alongside a clean chromatographic profile, since each method answers a question the other cannot.