DETERMINATION OF DISULFIDE BONDS IN HIGHLY BRIDGED DISULFIDE-LINKED PEPTIDES BY MATRIX-ASSISTED-LASER-DESORPTION IONIZATION MASS-SPECTROMETRY WITH POSTSOURCE DECAY/

Matrix-assisted laser desorption/ionization mass spectrometry with pos tsource decay was used to generate fragment ions from peptide fragment s containing heteropeptides linked together by two disulfide bonds, Po stsource decay analysis of these peptide samples Generates a series of singly charged fragment ions that, in addition to the peptide sequenc e ions, provide useful information for assigning disulfide arrangement in highly bridged disulfide-linked peptides, The assignment was made possible by fragmentation at peptide,bonds between two Cys residues in a peptide that constitutes the highly bridged fragment, while retaini ng the disulfide linkage to the other peptide, Fragmentation using oth er types of instruments, such as quadrupole ion-trap mass spectrometry with collision-induced dissociation, usually did not generate such fr agment ions, Tile data obtained from postsource decay also provide fra gment ions derived from both symmetric and nonsymmetric cleavages of d isulfide bonds, The present method is a highly sensitive technique whi ch requires no further sample handling and should be complementary to other classical chemical methods, The method proved useful in facilita ting the assignment of disulfide structure in tumor necrosis factor bi nding protein (TNFbp), which contains 162 amino acids and 13 disulfide bonds (Jones, M.; et al, Biochemistry, in press). Postsource decay an alysis of large disulfide-containing peptides usually produces no frag mentation but generates a series of high-intensity ions derived from b oth symmetric and nonsymmetric cleavages of disulfide bonds.