ShortCommunication GustavoChemale 1 ArjanJ.vanRossum 2 JamesR.Jefferies 3 JohnBarrett 2 PeterM.Brophy 2 HenriqueB.Ferreira 1 ArnaldoZaha 1 1 Centro de Biotecnologia and Departamento de Biologia Molecular e Biotecnologia, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil 2 Institute of Biological Sciences, University of Wales, Aberystwyth, Wales Proteomicanalysisofthelarvalstageofthe parasite Echinococcus granulosus:Causativeagent ofcystichydatiddisease We describe the preparation of Echinococcus granulosus metacestode protein extracts for two-dimensional electrophoresis (2-DE). Protoscoleces and hydatid fluid were prepared by precipitation using trichloroacetic acid (TCA) to remove nonprotein contaminants. Compared to the untreated control, TCA precipitation improved the 2-DE gel profile of the protoscoleces proteins. Comparison of 2-DE gels from insoluble and soluble fractions of the protoscoleces protein extract showed that most proteins are insoluble after lysis by sonication. Host serum proteins, especially albumin and globulins, caused horizontal streaking problems on the hydatid fluid 2-DE gels due to their high content in this sample. Even after the preparation of a hydatid fluid parasite enriched fraction, the high amount of bovine serum albumin and globulins made para- site-specific proteins difficult to detect by 2-DE. Despite the absence of an E.granulo- sus genome sequencing or expressed sequence tag (EST) projects, it was possible to identify 15 prominent protein spots from a whole protein protoscoleces 2-DE gel by peptide mass fingerprinting. These include actins, tropomyosin, paramyosin, thiore- doxin reductase, antigen P-29, cyclophilin, and the heat shock proteins hsp70 and hsp20. This work demonstrates that 2-DE and PMF are important tools to identify pro- teins from the hydatid fluid and protoscoleces and for the comparative analysis of cysts from different hosts or between active and resting cysts. Keywords: Actin / Echinococcus granulosus / Heat shock protein / Trichloracetic acid precipita- tion / Two-dimensional gel electrophoresis PRO 0487 Cystic hydatid disease (CHD) is an endemic helminthic disease caused by infection with the larval stage or meta- cestode of the tapeworm Echinococcus granulosus. The adult worm develops in the small intestine of definitive hosts, mainly dogs and other canids. The metacestode or hydatid cyst develops mostly in the liver and lungs of intermediate hosts, affecting humans and a wide range of livestock species [1]. The fully developed hydatid cyst of E. granulosus is unilocular and fluid (hydatid fluid, HF) filled. It consists of an inner germinal layer supported externally by a noncellular laminated layer, which is, in turn, surrounded externally by host-produced fibrous adventicial tissue. Protoscoleces (PSC), produced by asexual reproduction by the germinal layer, develop in the adult worm when ingested by the definitive host. E. granulosus occurs worldwide and CHD is recognized as one of the world’s major zoonoses [2]. Identification and characterization of proteins from E. granulosus metacestode might help to find new candi- dates for the immunodiagnosis and vaccines. Proteomics offers a set of tools for investigating proteins from para- sites, even those without a genome project [3]. It has the potential to identify proteins differentially expressed in dif- ferent parasite stages or in response to drugs [3], and excretory-secretory (EIS) products [4]. Even with the recent improvements and general protocols available, the analysis of protein samples by 2-DE requires stan- dardization for every tissue or organism under study [5]. In this paper, we describe for the first time the analysis of E. granulosus metacestode protein extracts by 2-DE and the identification of prominent proteins from a PSC 2-DE gel by peptide mass fingerprinting (PMF). PSC and HF were collected from hydatid cysts obtained from livers of cattle slaughtered at abattoirs in the Rio Grande do Sul State, southern Brazil. Elimination of non-protein content after TCA precipitation provided superior resolution of the Correspondence: Dr. Arnaldo Zaha, Centro de Biotecnologia and Departamento de Biologia Molecular e Biotecnologia, Uni- versidade Federal do Rio Grande do Sul, Caixa Postal 15005, Porto Alegre, 91501-970, RS, Brazil E-mail: zaha@dna.cbiot.ufrgs.br Fax: 155-51-33167309 Abbreviations: CHD, cystic hydatid disease; HF , hydatid fluid; PMF , peptide mass fingerprinting; PSC, protoscoleces Proteomics 2003, 3, 1633–1636 1633 DOI 10.1002/pmic.200300487  2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim