A robust, streamlined, and reproducible method for proteomic analysis of serum by delipidation, albumin and IgG depletion, and two-dimensional gel electrophoresis
A robust, streamlined, and reproducible method for proteomic analysis of serum by delipidation, albumin and IgG depletion, and two-dimensional gel electrophoresis
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DOI：
10.1002/pmic.200402048
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发表时间：
2005-07-01
期刊：
影响因子：
3.4
通讯作者：
Van Eyk, JE
中科院分区：
文献类型：
--
作者：
Fu, Q;Garnham, CP;Van Eyk, JE
血清是一种易于获得的诊断测定来源，但疾病特异性血清生物标志物的鉴定受到血清蛋白质组中人血清白蛋白和免疫球蛋白（Ig）占主导地位的阻碍。需要减少血清处理和分析中的技术变化，以允许对大型队列进行可重现的分析。为此，我们开发了一种快速和可重复的样品制备和高分辨率二维凝胶电泳分析人血清的程序。将血清高速离心以除去脂质和聚集的蛋白质，用蛋白G树脂孵育以除去IgG，用NaCl/乙醇沉淀以耗尽白蛋白，并在十二烷基硫酸钠（SDS）/N-（2-羟乙基）哌嗪-2'-（2-乙磺酸）（HEPES）缓冲液中缓慢再溶解。脱脂和IgG/alburnin耗尽的血清蛋白集中在pH 4 - 7线性大的固定化pH梯度条上，然后通过Bis-Tris SDS-聚丙烯酰胺凝胶电泳解析。连续三天测定三份个体血清样品的优化方法的耐用性和重现性。九个银染凝胶的图像分析表明，蛋白质斑点的强度和定位是高度可重复的。我们的IgG和白蛋白消耗程序将有助于筛选患者血清的正常生物学变异和疾病特异性生物标志物。
Serum is a readily available source for diagnostic assays, but the identification of disease-specific serum biomarkers has been impeded by the dominance of human serum albumin and immunoglobulins (Igs) in the serum proteome. There is a need to reduce the technical variation in serum processing and analysis to allow for a reproducible analysis of large cohorts. To this end, we have developed a rapid and reproducible procedure for sample preparation and high-resolution two-dimensional gel electrophoresis to analyze human serum. Serum is centrifuged at high speed to remove lipids and aggregated proteins, incubated with protein G resin to remove IgG, precipitated with NaCl/ethanol to deplete albumin, and slowly resolubilized in a sodium dodecyl sulfate (SDS)/N-(2-hydroxyethyl)piperazine-2'-(2-ethanesulfonic acid) (HEPES) buffer. The delipidated and IgG/alburnin depleted serum proteins are focused on pH 4-7 linear large immobilized pH gradient strips, and then resolved by Bis-Tris SDS-polyacrylamide gel electrophoresis. The robustness and reproducibility of the optimized procedure was determined for three individual serum samples on three consecutive days. An image analysis of the nine silver-stained gels demonstrated that the intensity and localization of protein spots are highly reproducible. Our IgG and albumin depletion procedure will aid in screening the patient sera for normal biological variation and disease-specific biomarkers.