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      Quantitative Proteomic Analysis of Venoms from Russian Vipers of Pelias Group: Phospholipases A 2 are the Main Venom Components

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          Abstract

          Venoms of most Russian viper species are poorly characterized. Here, by quantitative chromato-mass-spectrometry, we analyzed protein and peptide compositions of venoms from four Vipera species ( V. kaznakovi, V. renardi, V. orlovi and V. nikolskii) inhabiting different regions of Russia. In all these species, the main components were phospholipases A 2, their content ranging from 24% in V. orlovi to 65% in V. nikolskii. Altogether, enzyme content in venom of V. nikolskii reached ~85%. Among the non-enzymatic proteins, the most abundant were disintegrins (14%) in the V. renardi venom, C-type lectin like (12.5%) in V. kaznakovi, cysteine-rich venom proteins (12%) in V. orlovi and venom endothelial growth factors (8%) in V. nikolskii. In total, 210 proteins and 512 endogenous peptides were identified in the four viper venoms. They represented 14 snake venom protein families, most of which were found in the venoms of Vipera snakes previously. However, phospholipase B and nucleotide degrading enzymes were reported here for the first time. Compositions of V. kaznakovi and V. orlovi venoms were described for the first time and showed the greatest similarity among the four venoms studied, which probably reflected close relationship between these species within the “kaznakovi” complex.

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          Most cited references30

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          A practical guide to the MaxQuant computational platform for SILAC-based quantitative proteomics.

          MaxQuant is a quantitative proteomics software package designed for analyzing large mass spectrometric data sets. It is specifically aimed at high-resolution mass spectrometry (MS) data. Currently, Thermo LTQ-Orbitrap and LTQ-FT-ICR instruments are supported and Mascot is used as a search engine. This protocol explains step by step how to use MaxQuant on stable isotope labeling by amino acids in cell culture (SILAC) data obtained with double or triple labeling. Complex experimental designs, such as time series and drug-response data, are supported. A standard desktop computer is sufficient to fulfill the computational requirements. The workflow has been stress tested with more than 1,000 liquid chromatography/mass spectrometry runs in a single project. In a typical SILAC proteome experiment, hundreds of thousands of peptides and thousands of proteins are automatically and reliably quantified. Additional information for identified proteins, such as Gene Ontology, domain composition and pathway membership, is provided in the output tables ready for further bioinformatics analysis. The software is freely available at the MaxQuant home page.
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            Implementation and evaluation of relative and absolute quantification in shotgun proteomics with label-free methods.

            Tandem mass spectrometry allows for fast protein identification in a complex sample. As mass spectrometers get faster, more sensitive and more accurate, methods were devised by many academic research groups and commercial suppliers that allow protein research also in quantitative respect. Since label-free methods are an attractive alternative to labeling approaches for proteomics researchers seeking for accurate quantitative results we evaluated several open-source analysis tools in terms of performance on two reference data sets, explicitly generated for this purpose. In this paper we present an implementation, T3PQ (Top 3 Protein Quantification), of the method suggested by Silva and colleagues for LC-MS(E) applications and we demonstrate its applicability to data generated on FT-ICR instruments acquiring in data dependent acquisition (DDA) mode. In order to validate this method and to show its usefulness also for absolute protein quantification, we generated a reference data set of a sample containing four different proteins with known concentrations. Furthermore, we compare three other label-free quantification methods using a complex biological sample spiked with a standard protein in defined concentrations. We evaluate the applicability of these methods and the quality of the results in terms of robustness and dynamic range of the spiked-in protein as well as other proteins also detected in the mixture. We discuss drawbacks of each method individually and consider crucial points for experimental designs. The source code of our implementation is available under the terms of the GNU GPLv3 and can be downloaded from sourceforge (http://fqms.svn.sourceforge.net/svnroot/fqms). A tarball containing the data used for the evaluation is available on the FGCZ web server (http://fgcz-data.uzh.ch/public/T3PQ.tgz). (c) 2010 Elsevier B.V. All rights reserved.
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              Isolation and characterization of an apoptotic and platelet aggregation inhibiting L-amino acid oxidase from Vipera berus berus (common viper) venom.

              An L-amino acid oxidase was isolated from the venom of the common viper Vipera berus berus by a three-step procedure combining gel filtration, ion exchange and hydrophobic chromatography. The enzyme is a non-covalently bound homodimer with a monomeric molecular mass of 57.7 kDa. The N-terminal amino acid sequence and the internal peptide sequences show close structural homology with other snake venom L-amino acid oxidases. The purified protein catalyzed oxidative desamination of L-amino acids, the most specific substrate is L-Phe. The best substrates among the studied 20 amino acids were: L-Met, L-Leu, L-Phe, L-Ile, L-Arg and L-His. Five amino acids, L-Ser, L-Pro, Gly, L-Thr and L-Cys, were not oxidized. The enzyme inhibited ADP-induced platelet aggregation dose-dependently with an IC50 of 0.07 microM. The effect was neutralized by catalase. V. berus berus LAAO induced apoptosis in cultured HeLa and K562 cells as shown by DNA fragmentation gel pattern. The induction of apoptosis was inhibited by catalase.
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                Author and article information

                Contributors
                Role: Academic Editor
                Journal
                Toxins (Basel)
                Toxins (Basel)
                toxins
                Toxins
                MDPI
                2072-6651
                12 April 2016
                April 2016
                : 8
                : 4
                : 105
                Affiliations
                Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow 117997, Russia; xerx222@ 123456gmail.com (S.I.K.); rustam.ziganshin@ 123456gmail.com (R.H.Z.); vladislavstarkov@ 123456mail.ru (V.G.S.); vits@ 123456mx.ibch.ru (V.I.T.)
                Author notes
                [* ]Correspondence: utkin@ 123456mx.ibch.ru ; Tel.: +7-495-336-6522
                Article
                toxins-08-00105
                10.3390/toxins8040105
                4848631
                27077884
                65e91881-57da-42fc-8abb-84a94f32c5f9
                © 2016 by the authors; licensee MDPI, Basel, Switzerland.

                This article is an open access article distributed under the terms and conditions of the Creative Commons by Attribution (CC-BY) license ( http://creativecommons.org/licenses/by/4.0/).

                History
                : 15 February 2016
                : 05 April 2016
                Categories
                Article

                Molecular medicine
                snake venom,viper,vipera kaznakovi,vipera nikolskii,vipera orlovi,vipera renardi,proteome,mass-spectrometry

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