Studying disulfide bond rearrangement by MALDI-RTOF PSD and MALDI-TOF/RTOF high-energy CID (20 keV) experiments of peptides derived from ammodytoxins.
Ammodytoxins (Atxs) are presynaptically neurotoxic phospholipases present in Vipera ammodytes ammodytes snake venom. Atxs show a high sequence homology and contain 14 cysteines which form seven biologically relevant disulfide bridges-connecting non-neighboring cysteines. Formic acid cleavage was performed to confirm protein sequences by MALDI RTOF MS and resulted in 95.6% sequence coverage exhibiting only few formylations. Cysteine-containing peptides showed adjacent signals 2 and/or 4 Da lower (according to the number of cysteines present in the peptide) than the theoretical molecular weight indicating disulfide bridge rearrangement. Post-source decay (PSD) and high-energy collision-induced dissociation (CID) at 20 keV experiments showed fragmentation pattern unique for the reduced, thiol group containing and the oxidized, disulfide bridge harboring peptides. Besides typical low-energy fragment ions observed during PSD experiments (a-, b-, y-type ions), additional high-energy fragment ions (c-, x-, w-, d-type and internal fragments) of significant intensity were generated during fragmentation at 20 keV. In the case of charge directing N- and C-termini, x- and w-type ions were also observed during PSD. Good and up to complete sequence coverage was achieved for all studied peptides from Atxs in the case of high-energy CID, whereas PSD lacked information particularly for larger peptides.
Institute of Immunology, Zagreb, Croatia.
Marija
M
Brgles
Tihana
T
Kurtović
Beata
B
Halassy
Günter
G
Allmaier
Martina
M
Marchetti-Deschmann
D000595:Amino Acid Sequence; D004220:Disulfides / Q000737:chemistry*; D005561:Formates / Q000737:chemistry; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry*; D016415:Sequence Alignment; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D014757:Viper Venoms / Q000737:chemistry*
D004220:Disulfides; D005561:Formates; D010446:Peptide Fragments; D014757:Viper Venoms; C030544:formic acid