Research Article The Effect of Breed, Gender, and Acid Stimulation in Dog Saliva Proteome Sónia Lucena, 1,2 Ana V. Coelho , 3 Fernando Capela-Silva , 1,4 Asta Tvarijonaviciute , 5 and Elsa Lamy 1 1 Instituto de Ciˆ encias Agr´ arias e Ambientais Mediterrˆ anicas (ICAAM), Universidade de ´ Evora, 7000-083 ´ Evora, Portugal 2 Departamento de Medicina Veterin´ aria, Escola de Ciˆ encias e Tecnologia, Universidade de ´ Evora, 7000-083 ´ Evora, Portugal 3 Instituto de Tecnologia Qu´ ımica e Biol´ ogica Ant´ onio Xavier, Universidade Nova de Lisboa, 2780-157 Oeiras, Portugal 4 Departamento de Biologia, Escola de Ciˆ encias e Tecnologia, Universidade de ´ Evora, 7000-671 ´ Evora, Portugal 5 Interdisciplinary Laboratory of Clinical Analysis (Interlab-UMU), Regional Campus of International Excellence “Campus Mare Nostrum”, University of Murcia, 30100 Espinardo, Murcia, Spain Correspondence should be addressed to Elsa Lamy; ecsl@uevora.pt Received 10 February 2018; Accepted 4 April 2018; Published 3 June 2018 Academic Editor: Giuseppe Piccione Copyright © 2018 S´ onia Lucena et al. Tis is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Saliva gained interest as a potential noninvasive source of biomarkers in humans and that interest starts to be extended also to other animal species. For this purpose, the knowledge of the salivary proteome in healthy conditions and the factors that afect it and how they afect it are necessary. Te aim of the present study was to assess the efect that gender and breed have in saliva proteome and the changes in it induced by stimulation with acid. Saliva from 4 diferent purebred dogs (Portuguese Podengo, Greyhound, Rafeiro Alentejano, and Beagle) of both genders was collected without and afer stimulation with lemon juice. SDS-PAGE and two- dimensional gel electrophoresis (2-DE) profles were compared and the proteins of interest in-gel digested and identifed by mass spectrometry. Acid stimulation decreased total protein concentration and the relative amounts of some protein bands/spots. Gender appeared to have minimal efect in saliva proteome, whereas the infuence of breed varies. Beagles and Portuguese Podengos were the two breeds with higher diferences. In conclusion, stimulation procedures and dog breed should be considered in data analysis when using salivary proteins for diagnostic purposes. 1. Introduction Physiological variables are of added value to assess the welfare and lifespan both in humans and in animals, as they pro- vide important information for interpreting and validating emotional and biological responses, respectively [1]. Saliva has gained interest for biomarker identifcation, mainly due to the noninvasive nature of its collection; at the same time that it contains glandular and blood-born molecules that can change under diferent conditions [2]. In dogs, most of the studies have been focused on the evaluation of stress by measuring salivary cortisol levels [3]. Infectious agents, such as Helicobacter spp., Bartonella spp., or rabies virus, have also been evaluated [4–6]. In addition, canine saliva has been used for quantifcation of acute phase proteins [7] and allergen measurements [8] and in forensic studies for canine mRNA determination [9]. Furthermore, recently, healthy dog saliva proteome has been characterized by shotgun proteomics, with the identifcation of 2,491 proteins and peptides [10]. Despite this characterization, two-dimensional electrophoresis (2-DE) salivary protein profles of dog saliva have been less explored. Although several researchers con- sider that gel-based approaches provide limited information, 2-DE continues providing reliable quantitative results on diferential protein expressions as they display a high num- ber of protein species, their isoforms, and posttranslational modifcations at the same time [11]. It also has the advantage of allowing modifcations of the protein mixtures caused by inadequate treatment or endogenous protease activities with physiological relevance to be easily recognized via pattern disturbances by 2D gels [11]. Hindawi BioMed Research International Volume 2018, Article ID 7456894, 12 pages https://doi.org/10.1155/2018/7456894