Characterization of hapten binding to immunoconjugates by electrospray ionization mass spectrometry

Electrospray ionization mass spectrometry (ESI-MS) has been used to characterize the covalent binding of different haptens to the enzyme glucose 6-phosphate dehydrogenase. The technique allows one to directly observe the relative amounts of each conjugated species present in a mixture, as well as the average hapten number. These measurements are useful for optimizing reaction conditions to yield a more precisely defined product for use in immunoassays. The results obtained show that ESI-MS with a quadrupole analyzer can be successfully used to analyze mixtures of derivatized proteins in the molecular weight range of 50-60 kDa, where the modifying hapten has a molecular weight as low as 130 Da.