Rohde E, Tomlinson A J, Johnson D H, Naylor S
Biomedical Mass Spectrometry Facility, Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN 55905, USA.
J Chromatogr B Biomed Sci Appl. 1998 Aug 25;713(2):301-11. doi: 10.1016/s0378-4347(98)00209-6.
Fast and efficient analysis of proteins in physiological fluids is of great interest to researchers and clinicians alike. Capillary electrophoresis (CE) has proven to be a potentially valuable tool for the separation of proteins in specimens. However, a generally acknowledged drawback of this technique is the limited sample volumes which can be loaded onto the CE capillary which results in a poor concentration limit of detection. In addition, matrix components in samples may also interfere with separation and detection of analytes. Membrane preconcentration-CE (mPC-CE) has proved to be effective in overcoming these problems. In this report, we describe the systematic evaluation of parameters affecting on-line preconcentration/clean-up and separation of protein mixtures by mPC-CE. Method development was carried out with a standard mixture of proteins (lysozyme, myoglobin, carbonic anhydrase, and human serum albumin). First, using MALDI-TOF-MS, membrane materials with cation-exchange (R-SO3H) or hydrophobic (C2, C8, C18, SDB) characteristics were evaluated for their potential to retain proteins in mPC cartridges. Hydrophobic membranes were found most suitable for this application. Next, all mPC-CE analysis of protein samples were performed in polybrene coated capillaries and parameters affecting sample loading, washing and elution, such as the composition and volume of the elution solvent were investigated. Furthermore, to achieve optimal mPC-CE performance for the separation of protein mixtures parameters affecting postelution focusing and electrophoresis, including the composition of the background electrolyte and a trailing stacking buffer were varied. Optimal conditions for mPC-CE analysis of proteins using a C2 impregnated membrane preconcentration (mPC) cartridge were achieved with a background electrolyte of 5% acetic acid and 2 mM ammonium acetate, 60 nl of 80% acetonitrile in H2O as an elution solvent, and 60 nl of 0.5% ammonium hydroxide as a trailing stacking buffer. The developed method was used successfully to separate proteins in aqueous humor, which contains numerous proteins in a complex matrix of salts.
对研究人员和临床医生而言，快速高效地分析生理体液中的蛋白质都极具吸引力。毛细管电泳（CE）已被证明是分离样本中蛋白质的一种潜在有价值的工具。然而，该技术一个普遍公认的缺点是可加载到CE毛细管上的样本体积有限，这导致检测浓度限较低。此外，样本中的基质成分也可能干扰分析物的分离和检测。膜预浓缩-毛细管电泳（mPC-CE）已被证明能有效克服这些问题。在本报告中，我们描述了对影响mPC-CE在线预浓缩/净化及蛋白质混合物分离的参数进行的系统评估。方法开发使用了蛋白质标准混合物（溶菌酶、肌红蛋白、碳酸酐酶和人血清白蛋白）。首先，使用基质辅助激光解吸电离飞行时间质谱（MALDI-TOF-MS），评估具有阳离子交换（R-SO3H）或疏水（C2、C8、C18、SDB）特性的膜材料在mPC柱中保留蛋白质的潜力。发现疏水膜最适合此应用。接下来，在涂有聚乙烯亚胺的毛细管中对蛋白质样本进行所有mPC-CE分析，并研究影响进样、洗涤和洗脱的参数，如洗脱溶剂的组成和体积。此外，为实现蛋白质混合物分离的最佳mPC-CE性能，改变影响洗脱后聚焦和电泳的参数，包括背景电解质和拖尾堆积缓冲液的组成。使用C2浸渍膜预浓缩（mPC）柱进行蛋白质mPC-CE分析的最佳条件是：背景电解质为5%乙酸和2 mM乙酸铵，60 nl 80%乙腈水溶液作为洗脱溶剂，60 nl 0.5%氢氧化铵作为拖尾堆积缓冲液。所开发的方法成功用于分离房水中的蛋白质，房水在盐的复杂基质中含有多种蛋白质。
