BACKGROUND Unlike humans, there is currently no publicly available reference mass spectrometry-based circulating acellular proteome data for sheep, limiting the analysis and interpretation of a range of physiological changes and disease states. The objective of this study was to develop a robust and comprehensive method to characterise the circulating acellular proteome in ovine serum. METHODS Serum samples from healthy sheep were subjected to shotgun proteomic analysis using nano liquid chromatography nano electrospray ionisation tandem mass spectrometry (nanoLC-nanoESI-MS/MS) on a quadrupole time-of-flight instrument (TripleTOF® 5600+, SCIEX). Proteins were identified using ProteinPilot™ (SCIEX) and Mascot (Matrix Science) software based on a minimum of two unmodified highly scoring unique peptides per protein at a false discovery rate (FDR) of 1% software by searching a subset of the Universal Protein Resource Knowledgebase (UniProtKB) database (http://www.uniprot.org). PeptideShaker (CompOmics, VIB-UGent) searches were used to validate protein identifications from ProteinPilot™ and Mascot. RESULTS ProteinPilot™ and Mascot identified 245 and 379 protein groups (IDs), respectively, and PeptideShaker validated 133 protein IDs from the entire dataset. Since Mascot software is considered the industry standard and identified the most proteins, these were analysed using the Protein ANalysis THrough Evolutionary Relationships (PANTHER) classification tool revealing the association of 349 genes with 127 protein pathway hits. These data are available via ProteomeXchange with identifier PXD004989. CONCLUSIONS These results demonstrated for the first time the feasibility of characterising the ovine circulating acellular proteome using nanoLC-nanoESI-MS/MS. This peptide spectral data contributes to a protein library that can be used to identify a wide range of proteins in ovine serum.

中文翻译：

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使用基于 LC-MS/MS 的血清蛋白质组学分析来表征健康绵羊的循环非细胞蛋白质组。

背景技术与人类不同，目前没有公开的基于参考质谱的绵羊循环非细胞蛋白质组数据，限制了对一系列生理变化和疾病状态的分析和解释。本研究的目的是开发一种稳健且全面的方法来表征绵羊血清中循环非细胞蛋白质组。方法 使用四极杆飞行时间仪器 (TripleTOF® 5600+, SCIEX) 上的纳米液相色谱纳米电喷雾电离串联质谱 (nanoLC-nanoESI-MS/MS) 对健康羊的血清样品进行鸟枪法蛋白质组分析。使用 ProteinPilot™ (SCIEX) 和 Mascot (Matrix Science) 软件通过搜索通用蛋白质资源的子集，基于每个蛋白质至少两个未经修饰的高评分独特肽，以 1% 的错误发现率 (FDR) 软件来鉴定蛋白质知识库 (UniProtKB) 数据库 (http://www.uniprot.org)。PeptideShaker（CompOmics、VIB-UGent）搜索用于验证 ProteinPilot™ 和 Mascot 的蛋白质鉴定。结果 ProteinPilot™ 和 Mascot 分别鉴定了 245 个和 379 个蛋白质组 (ID)，PeptideShaker 验证了整个数据集中的 133 个蛋白质 ID。由于 Mascot 软件被认为是行业标准并识别了最多的蛋白质，因此使用蛋白质分析通过进化关系 (PANTHER) 分类工具对这些蛋白质进行了分析，揭示了 349 个基因与 127 个蛋白质途径命中的关联。这些数据可通过 ProteomeXchange 获得，标识符为 PXD004989。结论 这些结果首次证明了使用 nanoLC-nanoESI-MS/MS 表征绵羊循环非细胞蛋白质组的可行性。该肽光谱数据有助于建立一个蛋白质库，可用于识别绵羊血清中的多种蛋白质。