PDF(Pubmed)
Abstract：
The sensitivity and specificity of tandem mass spectrometers have made targeted proteomics the method of choice for the precise simultaneous measurement of many proteins in complex mixtures. Its application to the relative quantification of proteins in high-density lipoproteins (HDL) that have been purified from human plasma has revealed potential mechanisms to explain the atheroprotective effects of HDL. We describe a moderate throughput method for isolating HDL from human plasma that uses sequential density gradient ultracentrifugation, the traditional method of HDL purification, and subsequent trypsin digestion and nanoflow liquid chromatography-tandem mass spectrometry to quantify 38 proteins in the HDL fraction of human plasma. To control for the variability associated with digestion, matrix effects, and instrument performance, we normalize the signal from endogenous HDL protein-associated peptides liberated during trypsin digestion to the signal from peptides liberated from stable isotope-labeled apolipoprotein A-I spiked in as an internal standard prior to digestion. The method has good reproducibility and other desirable characteristics for preclinical research.
摘要：
串联质谱仪的灵敏度和特异性使靶向蛋白质组学成为精确同时测量复杂混合物中许多蛋白质的首选方法。它在从人血浆中纯化的高密度脂蛋白（HDL）中蛋白质的相对定量中的应用揭示了解释HDL的动脉粥样硬化保护作用的潜在机制。我们描述了一种中等通量的方法，用于从人血浆中分离HDL，使用顺序密度梯度超速离心，传统的HDL纯化方法，以及随后的胰蛋白酶消化和纳流液相色谱-串联质谱法来定量人血浆HDL组分中的38种蛋白质。为了控制与消化相关的变异性，矩阵效应，和仪器性能，我们将胰蛋白酶消化过程中释放的内源性HDL蛋白相关肽的信号归一化为消化前作为内标掺入的稳定同位素标记载脂蛋白A-I释放的肽的信号.该方法具有良好的可重复性和临床前研究的其他理想特征。
