Several problems in pharmacology will be addressed, in which significant progress can be made by determining the primary sequence of proteins or altered proteins. The speed and accuracy of mass spectrometry will be brought to these problems, in conjunction with chemical and enzymatic techniques. New mass spectrometry techniques will be developed in support of protein structure studies. We will continue studies directed at understanding the multiple mechanisms for the drug resistance often acquired during chemotherapy, by characterizing the covalent sequestration proposed for both induced metallothionein and induced glutathione-Stransferase. Alkylation of purified proteins will be characterized for three therapeutic mustards, which have a range of chemical reactivities. In addition, sites of covalent bonding by activated glucuronides on human serum albumin and on cytosolic glutathione-S-transferases will be defined. Identification of modified residues may provide new information about binding sites for these widely occurring metabolites, and provide a molecular mechanism for the idiopathic immunogenicity associated with nonsteroidal anti-inflammatory and other drugs. Polyethylene glycol-derivatized adenosine deaminase (PEG-ADA) is an orphan drug used in treatment of severe immunosuppression syndrome. Analytical methods will be developed to characterize this very complex mixture, to understand the structural determinants of its plasma stability and immunocompatibility, and to enable metabolism studies. These will be applicable to other PEGated proteins under development for therapeutic uses. In combination these studies will provide insight as to whether xenobiotic alkylation of serum and cytosolic proteins is random or ordered, and how structural features in the drug and protein influence selectivity. Mass spectrometry techniques under development in support of these studies of pharmacologically relevant proteins include reaction induced dissociation in our high performance tandem mass spectrometer, and electrospray ionization with collisional activation or reaction induced dissociation.
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