Electrospray Mass Spectrometry: How Peptide Identity Is Confirmed

A chromatographic purity value does not, on its own, prove that the main component is the intended peptide. That question is answered by an identity test, and for peptides the most common one is mass spectrometry (MS).

The problem electrospray solved

Mass spectrometers measure ions in the gas phase. Large, polar, non-volatile molecules such as peptides and proteins are difficult to transfer into the gas phase intact. In 1989, Fenn, Mann, Meng, Wong and Whitehouse described Electrospray Ionization for Mass Spectrometry of Large Biomolecules in Science. In electrospray ionisation (ESI), a solution is sprayed through a charged capillary; as droplets evaporate, intact molecules are released as ions.

Multiple charges

A key feature reported in that work is that large molecules acquire multiple charges. Because a mass spectrometer measures mass-to-charge ratio, multiply charged ions appear within the measurable range of common instruments, and the molecular mass can be calculated from the series of charge states.

What this means for a COA

Electrospray is easily coupled to liquid chromatography (LC-MS), so the main HPLC peak can be checked against the expected molecular mass of the sequence. When reading analytical documentation, an identity result reported as an observed mass alongside the theoretical mass is more informative than a statement that identity "conforms" without data.