Direct and Indirect ESI-MS Analysis of Non-covalent Drug-Protein Complexes

Electrospray ionization mass spectrometry (ESI-MS) is a powerful technique for analyzing non-covalent complexes formed between small molecules and proteins.

Two ESI-MS approaches can be taken, namely, direct and indirect analysis of the complexes. Direct methods utilize exclusively ESI-MS to analyze the nature of the non-covalent complexes formed under native conditions in the condensed phase while analyzing the products in the gas phase. Indirect methods utilize biochemical and chromatographic methods for preparing and separating the complexes and ESI-MS as the ancillary detector for the individual products of the non-covalent complex, namely, the small molecules and the protein.

Direct analyses of non-covalent complexes between drug candidates and biopolymers have been studied extensively by ESI-MS. This subject has been reviewed comprehensively [2-10] and is also discussed in Chapter 10. The underlying principle of these ESI-MS studies is that the mass spectrometer directly analyzes, in the gas phase and in the absence of solvent, the complexes prepared in the condensed phase under native conditions, generally at a pH of @7 in water with a volatile buffer, most often ammonium acetate. Under these native conditions, the sensitivity of the ESI mass spectrometer is not optimum and there is no guarantee that the desolvated complex formed in the gas phase is not an artifact of the ion formation mechanism. In addition, the study of these complexes under native conditions is time-consuming because of the low sensitivity and difficulty in maintaining a stable instrument at the higher pressures needed to form and stabilize these protein complexes for mass spectral studies. Higher sensitivity is achieved under lower pH conditions and with more volatile solvents such as acetonitrile or methanol, however, these conditions denature the protein-drug complex.

A number of indirect methods have been developed with mass spectrometric detection to rapidly study non-covalent complexes for drug screening purposes [2]. Among the most promising and simple indirect methods that overcome the limitations described above for directly studying non-covalent complexes by mass spectrometry is the application of size exclusion techniques in the spin column format for the screening and analysis of drug-protein complexes under optimum mass spectral sensitivity conditions [11-13].

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