College of Chemistry, Beijing Normal University, No. 44, Beijing 100875, China.
J Chromatogr A. 2010 Jan 15;1217(3):405-10. doi: 10.1016/j.chroma.2009.11.056. Epub 2009 Nov 20.
To improve the detection sensitivity and determine phenotypes of haptoglobin (Hp), a prefilling technique was developed and tested in capillary electrophoresis (CE) with UV-vis absorbance detection. Adding 0.01% sodium dodecyl sulfate (SDS) to the protein sample and 0.1% SDS to the prefilling buffer solution, on-line stacking and microheterogeneity separation of Hp were achieved. In addition, the influences of pH, buffer concentration, sample and prefilling buffer SDS concentration upon resolution were examined. Under optimized conditions, Hp-microheterogeneity was well resolved and two phenotypes of Hp (Hp 1-1 and Hp 2-2) were differentiated. This method was applied to the analysis of sera from normal individuals and beta-Thalassemia patients. After the depletion of albumin (HSA) and immunoglobulin G (IgG), this method allowed to determine two phenotypes in different individuals and to detect the decrease of Hp in beta-Thalassemia patients. Featuring high efficiency, speed and simplicity, the proposed method shows great potential for use in clinical diagnosis and proteome research.
为了提高触珠蛋白 (Hp) 的检测灵敏度并确定其表型，本研究开发并测试了一种在毛细管电泳 (CE) 中结合紫外可见吸收检测的预填充技术。在蛋白质样品中添加 0.01%十二烷基硫酸钠 (SDS)，在预填充缓冲溶液中添加 0.1% SDS，实现了 Hp 的在线堆积和微异质性分离。此外，还考察了 pH 值、缓冲液浓度、样品和预填充缓冲液 SDS 浓度对分辨率的影响。在优化条件下，很好地分辨了 Hp 的微异质性，并区分了 Hp 的两种表型 (Hp 1-1 和 Hp 2-2)。该方法应用于正常个体和β-地中海贫血患者血清的分析。在耗尽白蛋白 (HSA) 和免疫球蛋白 G (IgG) 后，该方法可以在不同个体中确定两种表型，并检测到β-地中海贫血患者 Hp 的减少。该方法具有高效、快速和简单的特点，在临床诊断和蛋白质组学研究中具有很大的应用潜力。
