Electrospray ionisation mass spectrometry has now been extensively applied to the characterisation of biomolecules including peptides, proteins and oligonucleotides. ESI is a very gentle ionisation method with multiply-charged ions and minimal fragmentation generally observed in ESI mass spectra. There has also been considerable recent interest in the study of specific non-covalent associations of biomolecules by ESI [1]. The gentle nature of the ESI process means that many such associations known to exist in solution are preserved in the gas phase following ionisation.
We report here the results of experiments with a number of biological systems designed to provide a better understanding of the relationship between solution and gas phase non-covalent species. For example, we have used ESI-MS to examine the covalent binding of the antitumour agent hedamycin which both intercalates into and alkylates DNA, to self-complementary oligonucleotides. Results of titration experiments with hedamycin adducts and free oligonucleotides provide strong evidence that ions due to duplex forms observed in ESI mass spectra are the result of specific base-paired associations in the gas phase rather than non-specific interactions. We are also investigating the sequence dependence of oligonucleotide duplex formation.
The cyclic depsipeptide antibiotic valinomycin acts as an ion transporter across cell membranes and is known to exhibit a strong preference for K+ compared with Na+. This is reflected in ESI spectra of valinomycin when mixed with equimolar concentrations of K+ and Na+ ions, which contrasts with previous studies of this peptide using field desorption (FD) and fast atom bombardment (FAB).
We have also examined the non-covalent interactions of a range of proteins. For example: the binding of trypsin (24 kDa) to a serine protease inhibitor from the Australian native plant Sycios Australis has been shown by ESI to occur in a 1:1 molar ratio, consistent with solution binding studies of these inhibitors; and the interaction of the hydrophobic probe ANS with alpha-crystallin subunits has been demonstrated in ESI mass spectra.
Finally, the experimental parameters necessary to generate non-covalent adducts by ESI will be discussed and the strengths and limitations of using ESI for probing non-covalent interactions of biomolecules will be discussed.