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Proteomics of Multigenic Families from Species Underrepresented in Databases: The Case of Loquat (Eriobotrya japonica Lindl.) Polyphenol Oxidases Susana Selle ´s-Marchart, † Ignacio Luque, ‡ Juan Casado-Vela, § Maria Jose ´ Martı ńez-Esteso, † and Roque Bru-Martı ńez* ,† Grupo de Proteo ´mica y Geno ´mica Funcional de Plantas, Departamento de Agroquı ´mica y Bioquı ´mica, Facultad de Ciencias, Universidad de Alicante, Spain, Instituto de Bioquı ´mica Vegetal y Fotosı ńtesis (CSIC/ Universidad de Sevilla), Avenida Ame ´rico Vespucio 49.41092 Sevilla, Spain, Protein Technology Unit, Biotechnology Programme, Spanish National Cancer Centre (CNIO), Madrid, Spain Received October 23, 2007 Here, we approach the problem of obtaining accurate and reliable information about the gene origin of a protein belonging to a multigenic family, polyphenol oxidase, from an underrepresented species, Eriobotrya japonica. De novo sequencing was a key approach to obtain broad sequence coverage. Alignment of peptides on their most similar homologous protein revealed divergent amino acid positions that lead to hypothesize the minimal number of genes encoding for the proteins analyzed. Keywords: de novo sequencing • underrepresented species in databases • multigenic families • polyphenol oxidase • phylogenetic analysis 1. Introduction Protein identification is a central issue in proteomics that can be routinely achieved through the comparison of experimental mass spectral peak lists with theoretical ones calculated after a peptide database. 1,2 Real protein identification thus involves the assignment of experimental MS data from the protein to a gene product. Therefore, real identification can only be possible when the exact gene or protein sequence is present in the database. 3 Otherwise, the information from mass spectrometry (MS) data may merely help to discover the protein function by homology to sequences sharing peptides with the protein under study. Moreover, the criteria of sharing of peptides between orthologs may be not enough to assign a function to the protein, as proteins with different functions may share one peptide or more that have been referred to as degenerate peptides. 4 In the case of proteins encoded by a multigenic family of paralogous genes, the number of shared peptides is expected to increase, thus, making the real identification even more complex. Delanlade et al. 5 proposed an strategy to identify proteins at the paralog level by searching a genome database to genuinely identify a protein by means of discriminating peptides. Since only a few eukaryotic species are wide-genome-sequenced, this strategy has been relatively restricted to date. Nonetheless, analyzing and discovering the gene origin of a protein is a key milestone in the detailed understanding of the biological processes in which it functions, thus, enabling to address its relevance as a biomarker, a drug target, and so forth. Given the above rationale, it is obvious that the univocal identification of a protein from an underrepresented species in databases using standard approaches is an impossible enterprise. However, the combination of a series of proteomic and bioinformatic resources may lead to obtain quite accurate information about the gene origin of such a protein. Here, we deal with the case of polyphenol oxidase (PPO) from loquat (Eriobotrya japonica Lindl.) fruit, a Rosaceae family species to which other important tree fruit crops belong, such as apple, pear, peach, apricot, prune, almond, and so forth. Polyphenol oxidases (PPOs) (EC 1.14.18.1 or EC 1.10.3.2) are ubiquitous plant enzymes that catalyze the O-dependent oxidation of mono- and o-diphenols to o-quinones. The oxidation of phenolic substrates by PPO causes enzymatic browning of many fruits and vegetables during ripening as well as handling, storage and processing 6–9 which is a problem of considerable importance to the agri-food industry. 6,7 Multigenic families encoding different PPO polypeptides have been reported for tomato, 10 potato, 11 banana, 12 and hybrid poplar. 13 In Rosaceae, Southern experiments provide evidence of the existence of several PPO genes, 14 while two different PPO genes have been cloned and sequenced for apple. 15 The expression of PPO paralogs has been demon- strated to be regulated both developmentally and following a variety of stress conditions in a number of species, 11–13,15–18 although the coexpression of two PPO genes or more in the same tissue at the same developmental stage has also been reported. 15,16 Moreover, in apricot fruit, the PPO protein and its catalytic activity are present whatever the fruit age, although * To whom correspondence should be addressed. Prof. Dr. Roque Bru Martı ńez, Grupo de Proteo ´mica y Geno ´mica Funcional de Plantas, Depar- tamento de Agroquı ´mica y Bioquı ´mica, Facultad de Ciencias, Universidad de Alicante, Campus de San Vicente del Raspeig, Apdo. 99, E-03080 Alicante, Spain. Phone: +34 965903400. Fax: +34 965903880. E-mail: [email protected]. † Universidad de Alicante. ‡ Instituto de Bioquı ´mica Vegetal y Fotosı ńtesis (CSIC/Universidad de Sevilla). § Spanish National Cancer Centre (CNIO). 10.1021/pr700687c CCC: $40.75 2008 American Chemical Society Journal of Proteome Research 2008, 7, 4095–4106 4095 Published on Web 07/12/2008

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