Sanz-Nebot Victoria, Balaguer Elvira, Benavente Fernando, Neusüss Christian, Barbosa José
Analytical Chemistry Department, University of Barcelona, Barcelona, Spain.
Electrophoresis. 2007 Jun;28(12):1949-57. doi: 10.1002/elps.200600648.
Human transferrin (Tf) is a model glycoprotein for congenital disorders of glycosylation (CDG) diagnosis. In the last few years, new CE-UV methods for intact Tf glycoforms analysis have been developed using nonvolatile BGEs and organic modifiers. However, the use of these BGEs does not allow the coupling of these procedures with electrospray MS (ESI-MS). In this study, a new CE-UV separation method of Tf glycoforms is developed, using a double-layer stable coating and a volatile BGE based on ammonium acetate. The separation method is optimized using standard Tf and their potential is demonstrated applying the method to the analysis of sera Tf from healthy individuals and CDG patients. The CE-UV separation method has been coupled to ESI-MS detection. Main parameters such as sheath liquid composition are optimized in order to obtain a good sensitivity. The CE-ESI-MS method has also been used in serum samples obtaining the separation of the different proteins present in serum and partial separation of Tf glycoforms. Different mass spectra and deconvoluted molecular masses were obtained for each sialoform, allowing unequivocal glycoform identification.
人转铁蛋白（Tf）是用于诊断先天性糖基化障碍（CDG）的一种典型糖蛋白。在过去几年中，已开发出使用非挥发性背景电解质（BGE）和有机改性剂来分析完整Tf糖型的新型毛细管电泳 - 紫外（CE - UV）方法。然而，这些BGE的使用不允许将这些程序与电喷雾质谱（ESI - MS）联用。在本研究中，开发了一种用于Tf糖型的新型CE - UV分离方法，该方法使用双层稳定涂层和基于醋酸铵的挥发性BGE。使用标准Tf对分离方法进行了优化，并通过将该方法应用于分析健康个体和CDG患者的血清Tf来证明其潜力。CE - UV分离方法已与ESI - MS检测联用。为了获得良好的灵敏度，对诸如鞘液组成等主要参数进行了优化。CE - ESI - MS方法也已用于血清样品，实现了血清中不同蛋白质的分离以及Tf糖型的部分分离。每种唾液酸化形式都获得了不同的质谱图和解卷积分子量，从而能够明确鉴定糖型。
