RESEARCH ARTICLE Proteomic analysis of the venom from the fish eating coral snake Micrurus surinamensis: Novel toxins, their function and phylogeny Timoteo Olamendi-Portugal 1 * , CesarV. F. Batista 1 * , Rita Restano-Cassulini 1 , Victoria Pando 1 , Oscar Villa-Hernandez 1 , Alfonso Zavaleta-Martínez-Vargas 2 , Maria C. Salas-Arruz 2 , Ricardo C. Rodríguez de la Vega 1 , Baltazar Becerril 1 and Lourival D. Possani 1 1 Instituto de Biotecnologia, Universidad Nacional Autónoma de México, Avenida Universidad Cuernavaca, Morelos, Mexico 2 Departamento Académico de Bioquímica, Biología Molecular y Farmacología, Facultad de Ciencias y Filosofia, Universidad Peruana Cayetano Heredia, Lima, Peru The protein composition of the soluble venom from the South American fish-eating coral snake Micrurus surinamensis surinamensis, here abbreviated M. surinamensis, was separated by RP-HPLC and 2-DE, and their components were analyzed by automatic Edman degradation, MALDI-TOF and ESI-MS/MS. Approximately 100 different molecules were identified. Sixty- two components possess molecular masses between 6 and 8 kDa, are basically charged molecules, among which are cytotoxins and neurotoxins lethal to fish (Brachidanios rerio). Six new toxins (abbreviated Ms1–Ms5 and Ms11) were fully sequenced. Amino acid sequences similar to the enzymes phospholipase A2 and amino acid oxidase were identified. Over 20 additional peptides were identified by sequencing minor components of the HPLC separation and from 2-DE gels. A functional assessment of the physiological activity of the six toxins was also performed by patch clamp using muscular nicotinic acetylcholine receptor assays. Vari- able degrees of blockade were observed, most of them reversible. The structural and func- tional data obtained were used for phylogenetic analysis, providing information on some evolutionary aspects of the venom components of this snake. This contribution increases by a factor of two the total number of a-neurotoxins sequenced from the Micrurus genus in cur- rently available literature. Received: July 6, 2007 Revised: January 8, 2008 Accepted: January 9, 2008 Keywords: Acetylcholine receptor / Mass spectrometry / Micrurus / Snake / Toxin Proteomics 2008, 8, 1919–1932 1919 1 Introduction Coral snakes constitute a taxonomic group formed by more than 120 species and subspecies and belong to the Elapidae family that comprises three genera: Micruroides, Leptomi- crurus, and Micrurus. The genus Micrurus comprehend al- Correspondence: Professor Lourival D. Possani, Instituto de Bio- tecnología – UNAM, Av. Universidad, 2001, Col. Chamilpa, Cuer- navaca, Morelos – CP 62210, Mexico E-mail: possani@ibt.unam.mx Fax: 152-77-73-172388 Abbreviations: Ach, acetylcholine; Glu-C, glutamic-C; LAO, L- amino acid oxidase; Lys-C, lysine-C; nAchr, nicotinic acetylcho- line receptor; PLA2, phospholipase A2 * Both authors contributed equally to this work. DOI 10.1002/pmic.200700668  2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com