LOCATION OF DISULFIDE BONDS IN PROTEINS BY FABMS

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The interconversion of thiols and disulfides is important for many biological functions such as protein synthesis, neurotransmitter activity, and enzyme structure. To better understand these functions on the molecular level, it is important to know the locations of disulfide cross-linkages in proteins. All of the present methods for locating disulfide bonds use highly specific cleavage reactions to produce disulfide-bonded peptides. Since these cleavage reactions are often ineffective, the proposed investigation will focus on developing methods for locating disulfide bonds in proteins which cannot be cleaved with the commonly used highly specific cleavage reactions. Peptides produced from the partial acid hydrolysis of a model protein, hen egg-white lysozyme, will be fractionated by reversed-phase HPLC and identified from their molecular weights, which will be determined by fast atom bombardment mass spectrometry (FABMS). An electrochemical detector will be used to reveal which fractions contain disulfide-bonded peptides. Computer-assisted analysis of the molecular weight information will be used to relate the molecular weights to specific segments of the protein, and hence to locate the disulfide bonds. A similar approach is which a directly-coupled microbore HPLC FABMS system will be developed to identify disulfide-bonded peptides in the hydrolysate. This approach could reduce sample analysis to a single HPLC FABMS run. As these methods are developed, they will be used to locate disulfide linkages in biologically important proteins such as luteinizing hormone and tissue-type plasminogen activator. Leuteinizing hormone, a gonadotropin which is directly involved in testicular and ovarian functions, is of interest because of the difficulty in producing disulfide-bonded peptides which are diagnostic for each of the disulfide bonds in the native material. Tissue-type plasminogen activator is an example of a large protein which is presently produced via recombinant DNA technology.