Table_3_Characterization of the plasma proteome from healthy adult dogs.XLSX

IntroductionBloodwork is a widely used diagnostic tool in veterinary medicine, as diagnosis and therapeutic interventions often rely on blood biomarkers. However, biomarkers available in veterinary medicine often lack sensitivity or specificity. Mass spectrometry-based proteomics technology has been extensively used in the analysis of biological fluids. It offers excellent potential for a more comprehensive characterization of the plasma proteome in veterinary medicine.
MethodsIn this study, we aimed to identify and quantify plasma proteins in a cohort of healthy dogs and compare two techniques for depleting high-abundance plasma proteins to enable the detection of lower-abundance proteins via label-free quantification liquid chromatography-mass spectrometry. We utilized surplus lithium-heparin plasma from 30 healthy dogs, subdivided into five groups of pooled plasma from 6 randomly selected individuals each. Firstly, we used a commercial kit to deplete high-abundance plasma proteins. Secondly, we employed an in-house method to remove albumin using Blue-Sepharose.
Results and discussionAmong all the samples, some of the most abundant proteins identified were apolipoprotein A and B, albumin, alpha-2-macroglobulin, fibrinogen beta chain, fibronectin, complement C3, serotransferrin, and coagulation factor V. However, neither of the depletion techniques achieved significant depletion of highly abundant proteins. Despite this limitation, we could detect and quantify many clinically relevant proteins. Determining the healthy canine proteome is a crucial first step in establishing a reference proteome for canine plasma. After enrichment, this reference proteome can later be utilized to identify protein markers associated with different diseases, thereby contributing to the diagnosis and prognosis of various pathologies.

引言
血液检测是兽医学领域广泛应用的诊断手段，疾病诊断与治疗干预常依赖血液生物标志物。然而，当前兽医学可用的生物标志物往往存在灵敏度或特异性不足的问题。基于质谱的蛋白质组学技术已被广泛应用于生物体液分析，在兽医学中具备全面解析血浆蛋白质组的巨大潜力。

方法
本研究旨在对一批健康犬的血浆蛋白进行鉴定与定量，并对比两种血浆高丰度蛋白去除方法，以通过无标记定量液相色谱-质谱联用技术（label-free quantification liquid chromatography-mass spectrometry）实现低丰度蛋白的检测。本研究使用30只健康犬的剩余肝素锂血浆，将其按每6只随机选取个体的混合血浆分为5组。其一，采用商品化试剂盒去除血浆高丰度蛋白；其二，利用实验室自建方法通过蓝色琼脂糖凝胶（Blue-Sepharose）去除白蛋白。

结果与讨论
在所有样本中，鉴定出的部分丰度最高的蛋白包括载脂蛋白A、载脂蛋白B、白蛋白、α2-巨球蛋白、纤维蛋白原β链、纤连蛋白、补体C3、转铁蛋白以及凝血因子V。但两种蛋白去除方法均未实现高丰度蛋白的有效去除。尽管存在这一局限，本研究仍成功检测并定量了多种临床相关蛋白。构建健康犬血浆蛋白质组是建立犬血浆参考蛋白质组的关键第一步。后续可基于该参考蛋白质组，识别与不同疾病相关的蛋白标志物，从而为多种病症的诊断与预后提供支持。