Petra Lutter

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

A method for the analysis of the rViscumin heterodimer (recombinant mistletoe lectin) based on two‐dimensional (2‐D) polyacrylamide gel electrophoresis and mass spectrometry was developed and used for quality control concerning purity and homogeneity of the recombinant protein processed under Good Manufacturing Practice (GMP) conditions. A series of spots with different pI‐values in the pH‐gradient of both rViscumin A‐ and B‐chain were observed independently 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 spectrometry (MALDI‐MS), liquid chromatography‐electrospray ionization (LC‐ESI)‐MS and LC‐ESI‐tandem MS (MS/MS) after tryptic in‐gel digestion resulted in a sequence coverage of 92% for the A‐chain and 95% for the B‐chain. No molecular differences like common chemical or post‐translational modifications or nonenzymatic deamidation were found to cause the different charge values of the separated spots. Therefore, these protein spots were extracted from 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 pI‐values. We suggest that the observed charge variants of rViscumin are the result of conformational protein variants, existing in an equilibrium during sample preparation and/or isoelectric focusing and are not caused from microheterogeneity in the primary structure of rViscumin.

Application of Proteomics and Protein Analysis for Biomarker and Target Finding for Immunotherapy

Regulatory T-cells play a central role in the maintenance of the immunological balance and are powerful inhibitors of T-cell activation both in vivo and in vitro. The enhancement of suppressor-cell function might be a target for immunotherapeutic approaches for the treatment of immune-mediated diseases like multiple sclerosis and Crohns disease.The method of choice to elucidate the still unclear effector functions of regulatory T-cells is the differential proteome analyses performed with human and murine T-cell populations. To this end, whole-protein extracts of conventional and regulatory T-cells are separated by high-resolution two-dimensional gel electrophoresis according to Klose. The proteomes are analyzed by a 2DE gel image analysis software, ProteomWeaver. The protein spots that are found differentially expressed are picked from the gels and prepared for matrix-assisted laser desorption/ionization (MALDI) mass spectrometrical analysis automatically. The high-resolution 2DE-PAGE and the automated spot handling and protein identification allows one to rapidly find new potential candidate proteins that are of functional relevance for regulatory T-cells, to be used as targets for drug development or as biomarkers for research and diagnostic purposes.