Assignment of disulfide bonds in proteins by fast atom bombardment mass spectrometry
Fast atom bombardment mass spectrometry has proved to be a powerful technique for locating the disulfide bonds in proteins. Both applications and potential of the method have been investigated in this study. The disulfide bonds in three proteins, human antithrombin III, hen egg-white lysozyme and ovine luteinizing hormone $\beta$ subunit, have been studied by FABMS analysis.^ Human antithrombin III is a single chain glycoprotein with 432 amino acid residues and three disulfide bonds. The protein is readily cleaved into disulfide-containing peptides which are analyzed by FABMS. Based on the molecular weights of the disulfide-containing peptides and their corresponding constituent peptides, the locations of three disulfide bonds are assigned to 8-128, 21-95 and 247-430.^ A new method has been studied for locating disulfide bonds in proteins which can not be cleaved between half-cystine residues by enzymatic methods. Partial acid hydrolysis has been applied to a model protein, hen egg-white lysozyme, to produce disulfide-containing peptides. HPLC fractions of the hydrolysate are analyzed by FABMS before and after chemical reduction. Computer assisted analysis is then used to relate the molecular weights obtained from FAB mass spectra to specific segments of the protein from which the disulfide connectivities can be deduced. The method is successfully applied to hen egg-white lysozyme to locate the four disulfide bonds in this protein.^ The method of FABMS analysis is finally applied to ovine luteinizing hormone $\beta$ subunit, a glycoprotein with 119 amino acid residues and six disulfide bonds. The protein is cleaved by combined use of partial acid hydrolysis and proteolysis. Three of six disulfide bonds are assigned as 23-72, 26-110 and 34-57. The obstacles for locating disulfide bonds in this protein have been discussed.^ The results presented in this thesis show that FABMS is a very sensitive, rapid, selective and accurate method for locating the disulfide bonds in proteins. The requirements of disulfide-containing peptides having proper sizes and containing only one disulfide bond may become a limitation when protein is highly cross-linked and the half-cystinyl residues are located with proximity in protein sequence. ^
Major Professor: David L. Smith, Purdue University.