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.
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.
Legros
Christian
C
UMR CNRS 8587, Laboratoire Analyse et Environnement, Institut des Sciences, Université d'Evry Val-d'Essonne, Bd. François Mitterrand, 91024 Evry Cedex, France.
Célérier
Marie-Louise
ML
Guette
Catherine
C
D000595:Amino Acid Sequence; D004229:Dithiothreitol / Q000737:chemistry*; D008969:Molecular Sequence Data; D036103:Nanotechnology; D010084:Oxidation-Reduction; D010455:Peptides / Q000737:chemistry*; D010720:Phosphines / Q000737:chemistry*; D020539:Sequence Analysis, Protein; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D013111:Spider Venoms / Q000737:chemistry*
D004229:Dithiothreitol; D010455:Peptides; D010720:Phosphines; D013111:Spider Venoms; C080938:tris(2-carboxyethyl)phosphine