Investigation of charge variants of rViscumin by two-dimensional gel electrophoresis and mass spectrometry

A method for the analysis of the rViscumin heterodimer (recombinant mistlet oe lectin) based on two-dimensional (2-D) poiyacrylamide gel electrophoresi s and mass spectrometry was developed and used for quality control concerni ng purity and homogeneity of the recombinant protein processed under Good M anufacturing Practice (GMP) conditions. A series of spots with different p/ -values in the pH-gradient of both rViscumin A- and B-chain were observed i ndependently from the experimental conditions like urea concentration, heat treatment or the use of cysteine alkylating agents. Comparative studies of the major spots using matrix assisted laser desorption/ionization-mass spe ctrometry (MALDI-MS), liquid chromatography-electrospray ionization (LC-ESI )-MS and LC-ESI-tandem MS (MS/MS) after tryptic in-gel digestion resulted i n a sequence coverage of 92% for the A-chain and 95% for the B-chain. No mo lecular differences like common chemical or post-translational modification s or nonenzymatic deamidation were found to cause the different charge valu es of the separated spots. Therefore, these protein spots were extracted fr om the 2-D gel and separated again by 2-D gel electrophoresis (termed Re-2- DE). Each of the single spots tested in the Re-2-DE experiment split up in the same heterogeneous pattern concerning the pl-values. We suggest that th e observed charge variants of rViscumin are the result of conformational pr otein variants, existing in an equilibrium during sample preparation and/or isoelectric focusing and are not caused from microheterogeneity in the pri mary structure of rViscumin.