Innovative high resolution mass spectrometry applications in drug discovery

In the past, the main applications of mass spectrometry in the pharmaceutical field were mainly oriented towards structure elucidation and quantitative analysis of drug and metabolites in biological matrices. Since the advent in the 90’s of soft ion sources such as ESI and MALDI, allowing the analysis of biomacromolecules (biopolymers such as DNA, proteins, peptides and sugars), MS has found growing applications in the different steps of drug discovery and development processes, including target identification, hit identification and lead identification and optimization. In particular, ESI source is the workhorse for the mass spectrometric analysis of noncovalent bound protein complexes since it allows intact weakly bound complexes to be detected reflecting the nature of the interaction found in the condensed phase. Several applications of such techniques have been reported, the most popular being the identification of selective and high affinity ligands by fishing libraries or using fragment based approaches. In particular, ligands are easily identified on the basis of the MW of the protein adduct and their affinity established on the basis of their relative abundance. The strength of this technique lies in its inherent sensitivity (picomolar quantities of protein and ligand required), its speed (as low as 30 s for each analysis), and its ability to simultaneously determine binding stoichiometry and dissociation constants. A further improvement in studying non covalent interactions came about more recently with the advent of nano-ESI source and high resolution MS analyzers such as orbitrap. Several applications have been reported and we have successfully applied this technique to screen targeted libraries against proteins [1,2] and nucleic acid [3]. Moreover, the effect of ligand interaction on protein conformation can be evaluated by studying the distribution of multicharged ions recorded in non denaturating conditions as well as the protein aa involved in the ligand recognition by H/D exchange experiments. The most recent investigation indicates that MS is also an excellent method for determining binding strength of protein-protein interactions within noncovalent complexes. The substantial advantages of MS over other techniques (e.g., ITC) are sensitivity and the ability to provide information on composition, stoichiometry and subunit interactions of protein complexes. References [1] L. Regazzoni, L. Bertoletti, G. Vistoli et al., ChemMedChem, 2010, 5, 1015-1025 [2] L. Regazzoni, R. Colombo, L. Bertoletti et al., Anal Chim Acta, 2011 [3] F. Riccardi Sirtori, G. Aldini, M. Colombo et al., submitted