Institute of Chemical Technologies and Analytics, Vienna University of Technology, Vienna, Austria.
Rapid Commun Mass Spectrom. 2010 Mar;24(6):761-7. doi: 10.1002/rcm.4440.
Von Willebrand factor (VWF), an adhesive glycoprotein with an approximate molecular weight (MW) of the monomer of 260 kDa, circulates in human blood plasma as a series of multimers ranging in size up to 20.000 kDa; thus the determination of the accurate MW of the monomer is of great importance and due to its high MW quite challenging. In this study accurate MW determination of intact recombinant VWF monomer (rVWF) was performed with GEMMA (gas-phase electrophoretic mobility macromolecular analysis) and MALDI TOF MS (matrix-assisted laser desorption/ionization linear time-of-flight mass spectrometry). Three rVWF preparations with differing buffer systems and glycoprotein concentrations were analyzed. First investigations directed towards heterogeneity determination by means of capillary gel electrophoresis (CGE)-on-the-chip with a laser-induced fluorescence detector revealed two compounds (MW of 277 kDa (migration time 44.3 s) and 341 kDa (migration time 49.5 s)) present in each sample to varying extents, namely mature and pro-rVWF. MALDI MS analysis in the linear positive ion mode allowed the detection of mature rVWF with an exact MW of 256.1 kDa (+/-0.8%) and pro-rVWF with a MW of 349.8 kDa (+/-0.8%). Two samples containing pro-rVWF in very minor concentration resulted in GEMMA detection of the mature rVWF with a MW of 227.4 kDa (+/-2.5%), derived from the measured globular size of 10.9 nm. For one sample containing both rVWF species in almost equal concentrations no differentiation of the two species was possible with GEMMA. Due to its lower resolution only a peak representing a mixture of both species at 11.8 nm could be observed, yielding a MW of 298.8 kDa (+/-1.6%).
血管性血友病因子（VWF）是一种黏附糖蛋白，其单体的近似分子量（MW）为 260kDa，在人血浆中以一系列大小不一的多聚体形式循环，大小可达 20000kDa；因此，准确测定单体的 MW 非常重要，由于其 MW 较高，所以具有一定挑战性。在这项研究中，使用 GEMMA（气相电泳迁移率大分子分析）和 MALDI-TOF MS（基质辅助激光解吸/电离线性时间飞行质谱）对完整重组 VWF 单体（rVWF）进行了准确 MW 测定。分析了三种具有不同缓冲体系和糖蛋白浓度的 rVWF 制剂。首先，通过带有激光诱导荧光检测器的毛细管凝胶电泳（CGE）-on-the-chip 进行了异质性测定的初步研究，结果表明，每个样品中存在两种不同程度的化合物（MW 为 277kDa（迁移时间 44.3s）和 341kDa（迁移时间 49.5s）），即成熟型和前体 rVWF。线性正离子模式下的 MALDI-MS 分析允许检测到精确 MW 为 256.1kDa（+/-0.8%）的成熟 rVWF 和 MW 为 349.8kDa（+/-0.8%）的前体 rVWF。两个含有极少量前体 rVWF 的样品通过 GEMMA 检测到 MW 为 227.4kDa（+/-2.5%）的成熟 rVWF，这是由测量得到的 10.9nm 的球状大小得出的。对于一个含有两种 rVWF 物种的样品，由于分辨率较低，仅观察到代表两种物质混合物的峰，MW 为 298.8kDa（+/-1.6%）。
