Determination with matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry of the extensive disulfide bonding in tarantula venom peptide Psalmopeotoxin I
| Autor: | Denis Mestivier, Cyril Pimentel, Hervé Darbon, Dietmar Waidelich, Audrey Combes, Jean-Michel Camadro, Soo Jin Choi |
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| Přispěvatelé: | Institut Jacques Monod (IJM (UMR_7592)), Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), Architecture et fonction des macromolécules biologiques (AFMB), Institut National de la Recherche Agronomique (INRA)-Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), Applera Deutschland GmbH, Centre National de la Recherche Scientifique (CNRS)-Aix Marseille Université (AMU)-Institut National de la Recherche Agronomique (INRA), Centre National de la Recherche Scientifique (CNRS)-Université Paris Diderot - Paris 7 (UPD7) |
| Jazyk: | angličtina |
| Rok vydání: | 2009 |
| Předmět: |
Stereochemistry
Molecular Sequence Data Plasmodium falciparum Spider Venoms Peptide MESH: Amino Acid Sequence Tandem mass spectrometry Mass spectrometry 01 natural sciences Antimalarials 03 medical and health sciences Fragmentation (mass spectrometry) Tandem Mass Spectrometry Arachnida Escherichia coli MESH: Arachnida Animals Organic chemistry MESH: Animals MESH: Disulfides [SDV.BBM]Life Sciences [q-bio]/Biochemistry Molecular Biology Amino Acid Sequence Cysteine Disulfides Peptide sequence Spectroscopy MESH: Plasmodium falciparum 030304 developmental biology chemistry.chemical_classification 0303 health sciences MESH: Molecular Sequence Data MESH: Escherichia coli 010401 analytical chemistry MESH: Tandem Mass Spectrometry General Medicine MESH: Cysteine MESH: Antimalarials Atomic and Molecular Physics and Optics MESH: Spider Venoms 0104 chemical sciences Matrix-assisted laser desorption/ionization MESH: Mutagenesis Site-Directed chemistry MESH: Spectrometry Mass Matrix-Assisted Laser Desorption-Ionization Spectrometry Mass Matrix-Assisted Laser Desorption-Ionization Mutagenesis Site-Directed Inhibitor cystine knot Time-of-flight mass spectrometry |
| Zdroj: | European Journal of Mass Spectrometry European Journal of Mass Spectrometry, IM Publications, 2009, 15 (4), pp.517-29. ⟨10.1255/ejms.1000⟩ European Journal of Mass Spectrometry, 2009, 15 (4), pp.517-29. ⟨10.1255/ejms.1000⟩ |
| ISSN: | 1469-0667 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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