A comparison of nano-electrospray gas-phase electrophoretic mobility macromolecular analysis and matrix-assisted laser desorption/ionization linear time-of-flight mass spectrometry for the characterization of the recombinant coagulation glycoprotein von W

Abstract

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%).