Jack Throck Watson Research Interests

The major research theme in this laboratory involves the characterization of peptides and proteins by mass spectrometry with fast atom bombardment (FAB), electrospray ionization (ESI), and matrix assisted laser desorption ionization (MALDI). Unlike classical mass spectrometry involving electron ionization (EI), the desorption/ionization (D/I) techniques (FAB, ESI, and MALDI) provide little or no fragmentation, and thus no basis for deducing structural features in the molecule of interest. On the other hand, peptides and proteins are not amenable to analysis by EI because they have no significant vapor pressure, and thus must be analyzed by the DI techniques which yield only an indication of the molecular weight.

We develop and apply microchemical modifications of the analyte that can be interweaved with molecular weight determinations by D/I at various stages of controlled degradation of the analyte in an effort to obtain structural information.

Primary Structure:

By installing a functional group (triphenylphosphonium) containing a fixed charge at the N-terminus of a peptide, we can reliably generate a series of ions that facilitates recognition of the sequence of amino acids in peptides up to 15-20 residues. This procedure allows us to improve the success rate in sequencing peptides by FAB in conjunction with CAD-MS/MS, ESI with in-source fragmentation, or MALDI using post-source decay analysis.

Secondary Structure:

Determination of the connectivity of disulfide bonds in a peptide or protein is essential for complete characterization. Classical approaches to disulfide bond mapping involve the use of proteases which require a cleavage site in between the cysteines, a constraint that becomes quite serious as the cysteines lie close to one another, and impossible if the cysteines are adjacent in the sequence. Furthermore, the proteolytic approach frequently requires alkaline conditions under which disulfide bond exchange can occur during the digestion leading to likely artifact formation. We have developed a chemical approach to disulfide bond mapping that involves chemical cleavage on the N-terminal side of cyanylated cysteines to yield degradation products that can be analyzed by D/I for mass mapping of the peptide or protein. By combining this cyanylation approach with the technique of partial reduction of proteins containing more than one disulfide bond, we have demonstrated that it is possible to deduce the connectivity of cysteines involved in a given disulfide bond. Furthermore, we can cyanylate at low pH to avoid disulfide bond exchange.

Our cyanylation/mass mapping approach also is applicable to peptides and proteins involving adjacent cysteines in their primary structure. This novel mass mapping approach to disulfide bond analysis offers new hope to protein chemists studying tightly knotted proteins that are refractory to conventional methodology. Representative projects include characterizing the disulfide bonding structure of proteins involved in von Willebrand disease (bleeding disorder), the putative misfolded proteins involved in cataract formation, vasoendothelial growth factor which controls growth of new blood vessels in cardiovascular beds and in tumor formation, and in natural products containing small, but tightly knotted peptides that inhibit HIV proteases.

Protein Folding:

Studies of the refolding pathways of an unfolded denatured protein are not only of academic interest, but of practical value in the pharmaceutical preparation oligopeptides formed via recombinant techniques. Proteins containing cystines that are involved in disulfide bond formation during the refolding process have been widely studied, but there is controversy concerning the trapping of folding intermediates because of problems with disulfide bond exchange, etc. We have demonstrated in studies with human epidermal growth factor that our cyanylation methodology is applicable to the trapping of sulfhydryl-containing intermediates involved in the refolding of cysteine-containing proteins.

Our cyanylation methodology offers the advantages of trapping the folding intermediates in an acidic medium to avoid disulfide bond exchange. Cyanylation of the free sulfhydryls quenches any further folding of the intermediates, and initiates the analytical protocol for direct determination of the disulfide bonding pattern. We are presently studying the refolding of long R3 insulin-like growth factor.

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