Analysis of mixtures of closely related forms of bovine trypsin by electrospray ionization mass spectrometry: use of charge state distributions to resolve ions of the different forms

Electrospray ionization mass spectrometry has been utilized for the analysis of bovine trypsin. Commercial active bovine trypsin is comprised of a mixture of three closely related different forms: beta-, alpha-, and psi-trypsin with similar molecular masses (23293, 23311, and 23329 mass units respectively). Peak broadening caused by the natural isotopic abundance of atoms in the protein molecules results in overlap of the ion peaks from the different trypsin forms. Therefore, accurate determination of the molecular masses of these trypsins in a mixture is not straightforward. However, because the trypsin isoforms have different number of basic groups and different higher order structures, the different forms acquire different numbers of charges. Therefore, ions from the different trypsins appear in different parts of the mass spectrum allowing accurate determination of their molecular masses.