Novel Successive Multi-Ionic Layers for ultra-efficient, diffusion-limited separation of intact proteins by capillary electrophoresis and capillary electrophoresis-mass spectrometry – SMIL E

Capillary electrophoresis (CE) is becoming increasingly important for the analysis of (intact) proteins due to its selectivity and high efficiency. This is especially the case when coupled to mass spectrometry (MS) since more sensitive interfaces for the CE-MS coupling are becoming available, as well as MS capabilities for protein characterization have been improved significantly, such as top-down MS/MS fragmentation. Nevertheless, the separation efficiency of CE often does not reach at all the theoretical expected values due to unwanted protein – capillary interactions. To prevent these adsorption phenomena, coatings have been developed. So far, the so-called Successive Multi-Ionic Layers (SMIL) coatings are among the best, as recently developed in our labs. However, even the still present very low residual protein adsorptions strongly limit the CE separation efficiency for protein analysis. In this project, we propose the synthesis of new polycations (the outermost layer of the coating, which will interact with the protein) with best performances regarding (i) stability of the coating, (ii) absence of protein adsorption and (iii) adjustability of the electro-osmotic flow. The proposed strategy not only includes novel polycations with additional zwitterionic groups for anti-adsorptive properties, but also for the first time homogeneous polymers based on peptide functional groups. These new synthesized polyelectrolytes will be evaluated by CE separation of a set of model proteins using 5-layers SMIL coatings in combination with poly(L-lysine citramide) (PLCA) and poly(methacrylic acid) (PMA). The new coatings will be classified with respect to residual adsorption, separation efficiency, and electroosmotic mobility. The best coatings will be used for various applications of intact protein analysis by CE-MS in pharmaceutical and biological fields. This will include the characterization of the charge heterogeneity of biopharmaceutically relevant antibodies as well the analysis of complex protein mixtures from cell cultures and body fluids. In order to achieve high robustness and best sensitivity a nanosheath-liquid CE-MS interface will be modified for the high-flow conditions of these polycationic coatings. Overall, we expect to develop novel coatings for glass surfaces preventing protein adsorption leading to best CE and CE-MS performance as well as many other potential applications, where this is needed.