Müller Roland, Marchetti-Deschmann Martina, Elgass Helmut, Breiteneder Heimo, Kratzmeier Martin, Allmaier Günter
Institute of Chemical Technologies and Analytics, Vienna University of Technology, Vienna, Austria.
Electrophoresis. 2010 Dec;31(23-24):3850-62. doi: 10.1002/elps.201000282.
The molecular weights (MW) of seven (glyco)proteins, of which five were plasma-derived, with MWs higher than 200 kDa were determined with three techniques: CGE-on-a-chip, SDS-PAGE and MALDI-TOF-MS. While the analysis of medium to high MW proteins with SDS-PAGE was an already well-established technique, the usefulness of MALDI-TOF-MS for the exact MW determination of high mass proteins was only partly described in literature so far. CGE-on-a-chip is the newest of all three applied techniques and was so far not applicable. Therefore, it was not evaluated for high MW (glyco)proteins. All proteins were analyzed under nonreducing as well as reducing conditions. In this work, it was demonstrated that all three described techniques were capable of determining the MW of all high molecular weight (glyco)proteins. The noncommercial CGE-on-a-chip assay allowed for the first time the electrophoretic separation of proteins in the MW range from 14 to 1000 kDa. MW assignment was limited to 500 kDa in the case of SDS-PAGE and 660 kDa in the case of the high MW CGE-on-a-chip assay. With the proper matrix and sample preparation, analysis with a standard MALDI-TOF-MS provided accurate MWs for all high MW proteins up to 1 MDa.
采用三种技术测定了7种（糖）蛋白的分子量（MW），其中5种源自血浆，分子量高于200 kDa：芯片毛细管凝胶电泳（CGE-on-a-chip）、十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）和基质辅助激光解吸电离飞行时间质谱（MALDI-TOF-MS）。虽然用SDS-PAGE分析中高MW蛋白已是一项成熟的技术，但到目前为止，MALDI-TOF-MS用于精确测定高质量蛋白MW的实用性在文献中仅部分有所描述。芯片毛细管凝胶电泳是所有三种应用技术中最新的一种，到目前为止还不适用。因此，未对高MW（糖）蛋白进行评估。所有蛋白质均在非还原和还原条件下进行分析。在这项工作中，证明了所描述的所有三种技术都能够测定所有高分子量（糖）蛋白的MW。非商业性的芯片毛细管凝胶电泳分析首次实现了14至1000 kDa MW范围内蛋白质的电泳分离。对于SDS-PAGE，MW分配限制在500 kDa，对于高MW芯片毛细管凝胶电泳分析，MW分配限制在660 kDa。通过适当的基质和样品制备，使用标准的MALDI-TOF-MS分析可为高达1 MDa的所有高MW蛋白提供准确的MW。
