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 postsource decay was used to generate fragment ions from peptide fragments containing heteropeptides linked together by two disulfide bonds. Postsource decay analysis of these peptide samples generates a series of singly charged fragment ions that, in addition to the peptide sequence 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 retaining the disulfide linkage to the other peptide. Fragmentation using other types of instruments, such as quadrupole ion-trap mass spectrometry with collision-induced dissociation, usually did not generate such fragment ions. The data obtained from postsource decay also provide fragment ions derived from both symmetric and nonsymmetric cleavages of disulfide bonds. The present method is a highly sensitive technique which requires no further sample handling and should be complementary to other classical chemical methods. The method proved useful in facilitating the assignment of disulfide structure in tumor necrosis factor binding protein (TNFbp), which contains 162 amino acids and 13 disulfide bonds (Jones, M.; et al. Biochemistry, in press). Postsource decay analysis of large disulfide-containing peptides usually produces no fragmentation but generates a series of high-intensity ions derived from both symmetric and nonsymmetric cleavages of disulfide bonds.
Jones
M D
MD
Department of Protein Structure, Amgen Inc., Amgen Center, Thousand Oaks, California 91320-1789, USA.
Patterson
S D
SD
Lu
H S
HS
D004220:Disulfides / Q000737:chemistry*; D004926:Escherichia coli / Q000737:chemistry; D010455:Peptides / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D004220:Disulfides; D010455:Peptides