European Journal of Mass Spectrometry - Abstract of Determination with matrix-assisted laser desorption/ionization tandem time- of-flight mass spectrometry of the extensive disulfide bonding in tarantula venom peptide Psalmopeotoxin I

Audrey Combes,^a Soo Jin Choi,^a,b Cyril Pimentel,^c Hervé Darbon,^c Dietmar Waidelich,^d Denis Mestivier^e and Jean-Michel Camadro^a,*
^aProtein Engineering and Metabolic Control, Molecular and Cellular Pathology Program, Jacques Monod Institute, UMR7592,—CNRS—Université Paris Diderot, 15 rue Hélène Brion, 75205 Paris Cedex 13, France. E-mail: camadro@ijm.jussieu.fr
^bPresent address: Department of Food Science and Technology, Seoul Women’s University, Seoul 139-774, Republic of Korea
^cArchitecture et Fonction des Macromolécules Biologiques, UMR6098 CNRS - Universités d’Aix-Marseille I and II, Marseille, France
^dApplera Deutschland GmbH, Darmstadt, Germany
^eModelling in Integrative Biology, Molecular and Cellular Pathology Program, Jacques Monod Institute, UMR7592,—CNRS—Université Paris Diderot, Paris, France

Psalmopeotoxin I (PcFK1) is a 33-residue peptide isolated from the venom of the tarantula Psalmopoeus cambridgei. This peptide specifically inhibits the intra-erythrocyte stage of Plasmodium falciparum in vitro. It contains six cysteine residues forming three disulfide bridges and belongs to the superfamily of natural peptides containing the inhibitor cystine knot (ICK) fold. We produced the wild-type and mutated forms of the recombinant peptide to examine the mechanism of action of PcFK1. The purified toxins were consistently produced as two isobaric peptides (r-PcFK1-1 and r-PcFK1- 2) with different retention properties but identical anti-plasmodial biological activity. Comparison of ¹⁵N-NMR heteronuclear single quantum correlation spectra revealed that although rPcFK1-1 was highly structured, rPcFK1-2 does not have a stable three-dimensional structure. We used high-energy collision-induced fragmentation of the peptides with a matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometer to further investigate the structure of the native peptides in its natural form and produced in E. coli. The fragmentation spectra of the native peptides were very complex due to the occurrence in the spectrum of ions resulting from (1) cross-linking of fragments through a disulfide bridge and (2) asymmetric fragmentations of the disulfide bridges and (3) multiple neutral losses. The tandem mass spectrometry fragmentation pattern of r-PcFK1-1 was similar to that of the natural peptide isolated from crude venom, but r-PcFK1-2 had a clearly distinct fragmentation pattern, more closely resembling the fragmentation spectra of reduced and alkylated peptides. Observed ions could be attributed to specific fragments by comparing spectra between the wild-type and selected variants with point mutations (Y11W, R20T, Y26W, K28V). The disulfide connections in r-PcFK1-2 differed from those of the native peptide and showed a rare disulfide bridge between vicinal cysteine residues. The r-PcFK1_(R20T) variant showed a very limited fragmentation pattern when analyzed in positive mode but displayed much more fragmentation in negative mode pointing out the importance of the R20 residue in the fragmentation of PcFK1. Using the reductive matrix 1,5-diaminonaphtalene promoted strongly in source decay fragmentation of the peptides in MS mode. Our findings illustrated the critical role of the electronic environment around the central Cys₁₈–Cys₁₉ doublet in PcFK1 in internal fragmentation of the peptide.