LC-MALDI-TOF/TOF experiments on venoms: a powerful approach for de novo and Top-Down sequencing of new pharmacological tools

Poster (2007, October 11)

Analyse de venins animaux par LC-MALDI-TOF/TOF: une approche efficace pour le sequencage de novo et Top-Down de nouveaux outils pharmacologiques

Poster (2007, September 17)

Top-Down Proteomics using Matrix-Enhanced ISD

Conference (2007, June 05)

New method for characterizing highly disulfide-bridged peptides in complex mixtures: Application to toxin identification from crude venoms

in Journal of Proteome Research (2007), 6(8), 3216-3223

Animal venoms are highly complex mixtures that can contain many disulfide-bridged toxins. This work presents an LC-MALDI approach allowing (1) a rapid classification of toxins according to their number of ... [more ▼]

Animal venoms are highly complex mixtures that can contain many disulfide-bridged toxins. This work presents an LC-MALDI approach allowing (1) a rapid classification of toxins according to their number of disulfide bonds and (2) a rapid top-down sequencing of the toxins using a new MALDI matrix enhancing in-source decay (ISD). The crude venom is separated twice by LC: the fractions of the first separation are spotted on the MALDI matrix alpha-cyano-4-hydroxycinnamic acid (CHCA) and the others using 1,5-diaminonaphthalene (1,5-DAN). CHCA spots are more convenient for obtaining a precise mass fingerprint of a large number of peptides; however, the analysis of 1,5-DAN spots allows the number of disulfide bridges to be counted owing to their partial in-plume reduction by this particular matrix. Subsequently, the disulfide bonds of all peptides present in the crude venom were reduced by an excess of tris(carboxyethyl)phosphine before the LC separation and were subjected to the same analysis in CHCA and 1,5-DAN. Toxins were sequenced using a TOF/TOF analysis of metastable fragments from CHCA spots and ISD fragmentation from 1,5-DAN spots. Novel conotoxin sequences were found using this approach. The use of 1,5-DAN for ISID top-down sequencing is also illustrated for higher molecular weight toxins such as snake cardiotoxins and neurotoxins (>6500 Da), where sequence coverage >70% is obtained from the c-ion series. [less ▲]

Fourier transform mass spectrometry: A powerful tool for toxin analysis

in Toxicon (2006), 47(6), 715-726

The crude venom of Conus virgo was analyzed by Fourier transform mass spectrometry (FTMS) using both nano-electrospray ionization and MALDI. The analyses were performed directly on the crude venom ... [more ▼]

The crude venom of Conus virgo was analyzed by Fourier transform mass spectrometry (FTMS) using both nano-electrospray ionization and MALDI. The analyses were performed directly on the crude venom, without chromatographic separation. The mass fingerprinting of the venom yielded 64 distinct molecular masses in the range 500-4500 Da with two major components at 1328.5142 and 1358.5592 Da. To facilitate the de novo sequencing of these compounds, the disulfide bonds of all components were reduced for the whole venom. The mass accuracy, resolution and sensitivity provided by FTMS were necessary to complete the sequencing of the two new peptides named ViVA and ViVB, that turned out to be conotoxins belonging to the T-superfamily, with the disulfide framework V. The peptides shared 80% similarity and as often observed for this class of compound, they were highly post-translationally modified: amidated C-terminus, pyroglutamic acid residue at the N-terminus and two disulfide bonds. Complementary online nano-LC-nano-ESI-FTMS experiments were undertaken. Among the 130 molecular masses found in the coupling experiments, only 45 were common with those obtained in the direct approach, which means that 21 compounds observed by nano-ESI-FTMS were not detected. This clearly shows that some discriminations against some classes of compounds occur when a chromatographic step is used before mass spectrometry. [less ▲]

Characterization of Toxins within Crude Venoms by Combined Use of Fourier Transform Mass Spectrometry and Cloning

in Analytical Chemistry (2005), 77(20), 6630-6639

The standard analytical procedure for screening the proteomic profile of a venom often relies on an appropriate combination of sample extraction, electrophoresis, reversed- phase high-performance liquid ... [more ▼]

The standard analytical procedure for screening the proteomic profile of a venom often relies on an appropriate combination of sample extraction, electrophoresis, reversed- phase high-performance liquid chromatography, mass spectrometry, and Edman degradation. We present in this study a new approach for venom screening based on Fourier transform mass spectrometry (FTMS) analysis directly on the crude venom. The venom chosen is a unique sample from Atractaspis irregularis, a species never studied at the molecular level previously. This snake belongs to the Atractaspidae family that is known to produce highly toxic venoms containing endothelin-like peptides called sarafotoxins (SRTXs). Nanoelectrospray- FTMS spectrum of the crude venom allowed the identification of 60 distinct compounds with molecular masses from 600 to 14 000 Da, which would have been impossible without the resolution of this kind of instrument. De novo sequencing within the entire venom confirmed the sequences of two new families of sarafotoxins, whose precursors had been cloned, and allowed the characterization of a third one. One particularly interesting point was that the propolypeptides appeared processed not in one unique compound, but rather in different length molecules ranging from 15 for the shorter to 30 amino acids for the longer. Moreover, our results clearly establish that in the case of A. irregularis only one copy of mature sarafotoxin emerges from each precursor, which is a totally different organization in comparison of other precursors of SRTXs. [less ▲]