An unusual cleavage reaction of a peptide observed during dithiotreitol and tris(2-carboxyethyl)phosphine reduction: application to sequencing of HpTx2 spider toxin using nanospray tandem mass spectrometry |
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Authors: | Legros Christian Célérier Marie-Louise Guette Catherine |
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Institution: | UMR CNRS 8587, Laboratoire Analyse et Environnement, Institut des Sciences, Université d'Evry Val-d'Essonne, Bd. Fran?ois Mitterrand, 91024 Evry Cedex, France. |
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Abstract: | A recombinant peptidic spider toxin, HpTx2, was investigated directly by nanoelectrospray tandem mass spectrometry (MS/MS). This 30-residue toxin possesses a highly knotted structure with cystines arranged in close proximity. The low-energy collision-induced dissociation MS/MS spectrum of the M+4H](4+) ion permitted characterization of the C-terminal sequence of HpTx2 up to Cys(26) that is involved in a disulfide bridge. Chemical pre-treatment with DTT or TCEP was then investigated, and it was found that an unexpected cleavage reaction of HpTx2 gave two smaller peptides which were completely sequenced by MS/MS experiments using a Qq-TOF mass spectrometer. This unusual hydrolysis reaction facilitated the determination of the complete sequence of the HpTx2 toxin. |
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