Biotechnology and Bioengineering Center, Department of Physiology, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, USA.
Physiol Genomics. 2010 Feb 4;40(3):208-15. doi: 10.1152/physiolgenomics.00136.2009. Epub 2009 Nov 10.
The implication of the various lipoprotein classes in the development of atherosclerotic cardiovascular disease has served to focus a great deal of attention on these particles over the past half-century. Using knowledge gained by the sequencing of the human genome, recent research efforts have been directed toward the elucidation of the proteomes of several lipoprotein subclasses. One of the challenges of such proteomic experimentation is the ability to initially isolate plasma lipoproteins subsequent to their analysis by mass spectrometry. Although several methods for the isolation of plasma lipoproteins are available, the most commonly utilized techniques require large sample volumes and may cause destruction and dissociation of lipoprotein particle-associated proteins. Fast protein liquid chromatography (FPLC) is a nondenaturing technique that has been validated for the isolation of plasma lipoproteins from relatively small sample volumes. In this study, we present the use of FPLC in conjunction with nano-HPLC-ESI-tandem mass spectrometry as a new integrated methodology suitable for the proteomic analysis of human lipoprotein fractions. Results from our analysis show that only 200 microl of human plasma suffices for the isolation of whole high density lipoprotein (HDL) and the identification of the majority of all known HDL-associated proteins using mass spectrometry of the resulting fractions.
在过去的半个世纪中，各种脂蛋白类在动脉粥样硬化性心血管疾病发展中的意义使人们对这些颗粒给予了极大的关注。利用人类基因组测序所获得的知识，最近的研究工作已致力于阐明几种脂蛋白亚类的蛋白质组。此类蛋白质组实验的一个挑战是能够在通过质谱分析后最初分离血浆脂蛋白。尽管有几种分离血浆脂蛋白的方法，但最常用的技术需要大量的样本量，并且可能导致脂蛋白颗粒相关蛋白的破坏和解离。快速蛋白液相色谱（FPLC）是一种非变性技术，已通过从小体积的样本中分离血浆脂蛋白得到验证。在这项研究中，我们提出了将 FPLC 与纳升高效液相色谱-电喷雾串联质谱联用，作为一种新的集成方法，适用于人脂蛋白级分的蛋白质组学分析。我们的分析结果表明，仅用 200 微升人血浆即可用于分离整个高密度脂蛋白（HDL），并使用所得馏分的质谱法鉴定大多数已知的 HDL 相关蛋白。
