Escribano Damián, Horvatić Anita, Contreras-Aguilar Maria Dolores, Guillemin Nicolas, Cerón Jose Joaquín, Lopez-Arjona Marina, Hevia Maria Luisa, Eckersall Peter David, Manteca Xavier, Mrljak Vladimir
Department of Animal and Food Science, School of Veterinary Science, Universitat Autònoma de Barcelona, 08193 Cerdanyola del Vallès, Barcelona, Spain.
ERA Chair FP7, Internal Diseases Clinic, Faculty of Veterinary Medicine, University of Zagreb, Heinzelova 55, 10 000 Zagreb, Croatia.
Res Vet Sci. 2019 Jun;124:338-345. doi: 10.1016/j.rvsc.2019.04.012. Epub 2019 Apr 17.
The aim of this study was to identify biological pathways and proteins differentially expressed in the saliva proteome of sheep after the application of a model of stress, using high-resolution quantitative proteomics. In addition, one of the proteins differently expressed was verified and evaluated as a possible biomarker of stress in this species. Saliva paired samples from eight sheep before and after the application of a model of stress based on shearing were analysed using tandem mass tags (TMT). The TMT analysis allowed for the identification of new stress-related metabolic pathways and revealed 13 proteins, never described in saliva of sheep, that were differentially expressed between before and after the stress. Six of these proteins pertain to four major metabolic pathways affected, namely: canonical glycolysis, oxygen transport, neural nucleus development, and regulation of actin cytoskeleton reorganization. The rest of proteins were unmapped original proteins such as acyl-coenzyme-A-binding protein; complement C3; alpha-2-macroglobulin isoform-X1; type-II small proline-rich protein; lactoferrin; secretoglobin family-1D-member; and keratin, type-II cytoskeletal 6. Of these proteins, based on its biological significance and specific immunoassay availability, lactoferrin was selected for further validation. The immunoassay intra- and inter-assay coefficients of variation were lower than 13%. The method showed good linearity under dilution and recovery, and the detection limit was low enough to detect salivary lactoferrin levels. A significant decrease (P < 0.01) in salivary lactoferrin concentration in the sheep following the application of the model of stress was observed, suggesting that this protein could be a potential salivary biomarker of stress situations in sheep.
本研究的目的是通过高分辨率定量蛋白质组学，确定在施加应激模型后绵羊唾液蛋白质组中差异表达的生物途径和蛋白质。此外，对其中一种差异表达的蛋白质进行了验证和评估，以确定其是否可能作为该物种应激的生物标志物。使用串联质量标签（TMT）分析了八只绵羊在基于剪毛的应激模型应用前后的唾液配对样本。TMT分析有助于识别新的应激相关代谢途径，并揭示了13种在绵羊唾液中从未描述过的蛋白质，这些蛋白质在应激前后存在差异表达。其中六种蛋白质属于受影响的四个主要代谢途径，即：经典糖酵解、氧气运输、神经核发育和肌动蛋白细胞骨架重组的调节。其余蛋白质为未映射的原始蛋白质，如酰基辅酶A结合蛋白；补体C3；α-2-巨球蛋白异构体-X1；II型富含脯氨酸的小蛋白；乳铁蛋白；分泌球蛋白家族-1D-成员；以及II型细胞角蛋白6。基于这些蛋白质的生物学意义和特定免疫测定的可用性，选择乳铁蛋白进行进一步验证。免疫测定的批内和批间变异系数均低于13%。该方法在稀释和回收率方面表现出良好的线性，检测限低到足以检测唾液乳铁蛋白水平。观察到在应用应激模型后绵羊唾液乳铁蛋白浓度显著降低（P<0.01），这表明该蛋白质可能是绵羊应激状态的潜在唾液生物标志物。
