Analysis of human serum by liquid chromatography-mass spectrometry: Improved sample preparation and data analysis
Analysis of human serum by liquid chromatography-mass spectrometry: Improved sample preparation and data analysis
复制标题
DOI：
10.1016/j.chroma.2006.02.088
复制
发表时间：
2006-07-07
影响因子：
4.1
通讯作者：
Bischoff, R.
中科院分区：
文献类型：
--
作者：
Govorukhina, N. I.;Reijmers, T. H.;Bischoff, R.
生物标志物的发现是蛋白质组学研究中一个快速发展的领域。液相色谱-质谱联用技术（LC-MS）已成为血清等生物液体中蛋白质和多肽的灵敏检测、定量和鉴定的有力方法。然而，高丰度蛋白质的存在往往掩盖了低丰度蛋白质的存在，从而在蛋白质组学研究中通常阻碍了它们的检测和鉴定。为了对宫颈癌患者血清库的样本进行纵向和横断面的比较分析，已经开发了基于高丰度蛋白质消耗的方法，然后是胰蛋白酶消化和LC-MS。测试了两种样品制备方法消耗高丰度血清蛋白的效率，以及它们如何影响LC-MS数据集的可重复性。第一种方法包括在染料配体层析上消耗人血清白蛋白（HSA），在固定的蛋白a载体上消耗免疫球蛋白G (IgG)，然后进行胰蛋白酶消化，阳离子交换层析分离，在C18柱上捕获和反相LC-MS。第二种方法包括基于多重免疫亲和层析法去除6种最丰富的血清蛋白，然后进行胰蛋白酶消化，在c18柱上捕获，反相LC-MS。根据保留时间和峰面积对选定数量的内源性肽进行了总体程序的可重复性评估，结果表明，第二种方法除了耗时更少之外，可重复性更高（保留时间< 0.1% RSD，峰面积< 30% RSD）。应用LC-MS成分检测算法，结合主成分分析（PCA），可以将加钉马心脏细胞色素C的血清样品与未加钉的血清样品区分开来，并检测出加钉马心脏细胞色素C的浓度趋势，该趋势与2 μ l原始血清中加钉马心脏细胞色素C的量相关，达到5 pmol细胞色素C的水平。(c) 2006 Elsevier B.V.版权所有
Discovery of biomarkers is a fast developing field in proteomics research. Liquid chromatography coupled on line to mass spectrometry (LC-MS) has become a powerful method for the sensitive detection, quantification and identification of proteins and peptides in biological fluids like serum. However, the presence of highly abundant proteins often masks those of lower abundance and thus generally prevents their detection and identification in proteomics studies. To perform future comparative analyses of samples from a serum bank of cervical cancer patients in a longitudinal and cross-sectional manner, methodology based on the depletion of high-abundance proteins followed by tryptic digestion and LC-MS has been developed. Two sample preparation methods were tested in terms of their efficiency to deplete high-abundance serum proteins and how they affect the repeatability of the LC-MS data sets. The first method comprised depletion of human serum albumin (HSA) on a dye ligand chromatographic and immunoglobulin G (IgG) on an immobilized Protein A support followed by tryptic digestion, fractionation by cation-exchange chromatography, trapping on a C18 column and reversed-phase LC-MS. The second method included depletion of the six most abundant serum proteins based on multiple immunoaffinity chromatography followed by tryptic digestion, trapping on a C 18 column and reversed-phase LC-MS. Repeatability of the overall procedures was evaluated in terms of retention time and peak area for a selected number of endogenous peptides showing that the second method, besides being less time consuming, gave more repeatable results (retention time: < 0.1% RSD; peak area: < 30% RSD). Application of an LC-MS component detection algorithm followed by principal component analysis (PCA) enabled discrimination of serum samples that were spiked with horse heart cytochrome C from non-spiked serum and the detection of a concentration trend, which correlated to the amount of spiked horse heart cytochrome C to a level of 5 pmol cytochrome C in 2 mu l original serum. (c) 2006 Elsevier B.V. All rights reserved.