There are a number of operating parameters that must be carefully optimized and thoroughly understood if one is to analyze noncovalent complexes by mass spectrometry (MS). While it is always a good idea to have a known model system on which to optimize these parameters so that the "expected" complex is observed, it is equally important (if not more important) that the final operating parameters chosen are still able to distinguish specific complexation from nonspecific aggregation [1-5]. A carefully chosen (and biologically relevant) model system, along with buffers, ionization parameters, and desolvation conditions which maintain specific noncovalent complexes, and which do not produce nonspecific artifacts, is essential. As illustrated below, ion desolvation should be accomplished with minimal added energy to prevent unfolding and to minimize dissociation of the ligand from the complex ions. The choice of appropriate ion-ization mode is critical for both proteins and nucleic acids, since the location of charges is a function of solution and gas-phase pKa values. For example, many proteins can be ionized in positive or negative mode, but the choice of negative or positive ionization for generation of the most "native" form depends on the solution isoelectric point of the protein. Understanding the effect various experimental variables will have on the results of a known system can provide valuable insight into the applicability of the experimental parameters as applied to unknown systems. This is particularly important in the area of high throughput drug discovery where one clearly can not know a priori whether or not a given substrate-ligand pair is "supposed" to bind.
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