Quantitative and Selective Analysis of Feline Growth Related Proteins Using Parallel Reaction Monitoring High Resolution Mass Spectrometry

Today immunoassays are widely used in veterinary medicine, but lack of species specific assays often necessitates the use of assays developed for human applications. Mass spectrometry (MS) is an attractive alternative due to high specificity and versatility, allowing for species-independent analysis. Targeted MS-based quantification methods are valuable complements to large scale shotgun analysis. A method referred to as parallel reaction monitoring (PRM), implemented on Orbitrap MS, has lately been presented as an excellent alternative to more traditional selected reaction monitoring/multiple reaction monitoring (SRM/MRM) methods. The insulin-like growth factor (IGF)-system is not well described in the cat but there are indications of important differences between cats and humans. In feline medicine IGF–I is mainly analyzed for diagnosis of growth hormone disorders but also for research, while the other proteins in the IGF-system are not routinely analyzed within clinical practice. Here, a PRM method for quantification of IGF–I, IGF–II, IGF binding protein (BP) –3 and IGFBP–5 in feline serum is presented. Selective quantification was supported by the use of a newly launched internal standard named QPrEST™. Homology searches demonstrated the possibility to use this standard of human origin for quantification of the targeted feline proteins. Excellent quantitative sensitivity at the attomol/μL (pM) level and selectivity were obtained. As the presented approach is very generic we show that high resolution mass spectrometry in combination with PRM and QPrEST™ internal standards is a versatile tool for protein quantitation across multispecies.

当前，免疫测定法已在兽医学领域得到广泛应用，但由于缺乏物种特异性检测试剂盒，往往不得不使用针对人类开发的检测方法。质谱法（MS）凭借高特异性与通用性，成为极具吸引力的替代方案，可实现物种非依赖性分析。基于质谱的靶向定量方法是大规模鸟枪法蛋白质组分析的重要补充。近年来，一种在轨道阱质谱（Orbitrap MS）平台上实现的名为平行反应监测（PRM）的方法，被提出作为传统选择反应监测/多反应监测（SRM/MRM）方法的优质替代方案。胰岛素样生长因子（IGF）系统在猫中的研究尚不完善，但已有研究提示猫与人类的该系统存在重要差异。在猫科动物医学中，胰岛素样生长因子I（IGF-I）主要用于生长激素紊乱的诊断及相关研究，但胰岛素样生长因子系统中的其他蛋白尚未在临床实践中开展常规检测。本研究建立了一种可定量猫血清中IGF-I、IGF-II、胰岛素样生长因子结合蛋白（BP）-3及IGFBP-5的PRM方法。该方法采用新推出的内标QPrEST™实现选择性定量，同源性搜索结果证实，这款人源内标可用于目标猫源蛋白的定量分析。该方法在阿托摩尔/微升（皮摩尔浓度）级别展现出优异的定量灵敏度与选择性。鉴于该方法通用性极强，本研究证明，结合平行反应监测与QPrEST™内标的高分辨质谱法，是一种可实现跨物种蛋白质定量的通用工具。