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 Structures: 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, vasorendothelial 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 and entering into a collaboration with A. Robinson, Univ. of Delaware to study the refolding of the P22 tailspike protein, which involves the putative formation of disulfide bonds during the refolding process to a native structure that involves no disulfide bonds. The P22 tailspike protein is a good model for aggregation or misfolding of proteins, a process known to be the basis of various disease states such cystic fibrosis and Alzheimer’s disease. Better knowledge of intermediate structures involved in refolding, as well as in misfolding pathways, will provide the basis for rational design of inhibitors of misfolding pathways and of molecular chaperones to promote proper folding of oligopeptides.
Research Interests:
The J.T. Watson laboratory is currently devoted to the characterization of modifications to protein structure, in particular disulfide bond linkages and the formation of the secondary structures during the refolding of proteins. We have developed a chemical method for cleaving proteins on the N-terminal side of cysteines by cyanylating the protein and subjecting it to alkaline conditions to promote cleavage. Mass analysis of cleavage products from proteins of known sequence allow us to validate the sequence information available from cDNA sequencing and to deduce the connectivity involved in disulfide bonds. Our chemical cleavage approach is superior to the classical proteolytic approach to disulfide bond mapping in that our chemical approach avoids problems of disulfide bond scrambling and is applicable to proteins containing adjacent cysteines. We have found that it is possible to trap sulfhydryl-containing intermediates during the refolding of cysteine-containing proteins that involve disulfide bond formation in the native structure. "Snap shots" of the refolding process can be acquired by plunging aliquots of the folding solution into an acidic medium containing a cyanylating reagent; the acidic medium suppresses disulfide bond exchange and artifact formation, and cyanylation precludes any further folding of the protein and commences the analytical procedure toward characterizing the disulfide bond structure of the intermediate itself. We have demonstrated the applicability of our disulfide bond mapping procedure to a variety of proteins including ribonuclease A which contains four disulfide bonds; we have also used the cyanylation mass mapping procedure for trapping and identifying the folding intermediates of human epidermal growth factor and long insulin-like growth factor.
Representative Publications:
This page last updated January 5, 1999.