How it works
Electrospray ionization is a “soft” ionization method. A solution of the peptide is pushed through a needle held at high voltage, producing a fine mist of highly charged droplets; as the solvent evaporates the droplets shed charge and shrink until bare, intact, multiply-charged ions remain. “Soft” means the molecule is ionized without being fragmented, so its intact mass is preserved.
Peptides characteristically pick up more than one proton, giving a series of ions such as [M+H]⁺, [M+2H]²⁺, and [M+3H]³⁺. Each appears at a different m/z because the charge (z) in the denominator differs, so a single compound produces a ladder of related peaks rather than one, and the underlying neutral mass can be reconstructed from any two members of the series.
The analyser separates ions by m/z and the detector records their abundance, producing a mass spectrum — abundance plotted against m/z. Because the expected m/z values follow directly from the molecular formula, an observed peak matching the expected [M+H]⁺, within the instrument’s mass accuracy, confirms that the molecule present has the expected mass.
Two mass conventions matter when comparing values: the monoisotopic mass, calculated from the most abundant isotope of each element, and the average mass, weighted over natural isotope abundances. High-resolution instruments resolve the isotope pattern and are compared against the monoisotopic value; the two figures differ slightly and should not be mixed.
Why it matters
Mass spectrometry answers the identity question a purity method cannot: it indicates whether the molecule you have is the molecule you intended, independent of how much of it is present. A preparation can be highly pure by HPLC and still be the wrong compound, and ESI-MS is the check against that.
Because the expected masses are computed from the sequence, ESI-MS is effectively a pre-registered test — the target value is fixed before the measurement is made, so a match is meaningful rather than post-hoc. A deletion, an extra residue, or an unexpected modification shifts the observed mass in a predictable, diagnosable direction.
Identity and purity together — mass by ESI-MS and area percent by RP-HPLC — are the two independent measurements a characterization record is built on. Neither substitutes for the other.
At Lineará
Every compound page publishes the expected ESI-MS series for the molecule, and the certificates confirm identity by comparing the observed mass-to-charge ratio against that expected series.
The compound identifiers — molecular formula, monoisotopic mass, and expected m/z — are derived by construction and reconciled against an independently computed formula and mass, so the expected value a certificate is checked against is itself a checked value.