Chemical Mobilization-Based Capillary Isoelectric Focusing–Mass Spectrometry Using the nanoCEasy Interface for Pharmaceutical Protein Analysis

Capillary isoelectric focusing (CIEF) coupled with electrospray ionization mass spectrometry (ESI-MS) is regarded as an outstanding approach for protein and proteoform analysis, combining a high-resolution separation technique and an enhanced detection technique. The few so-far developed CIEF–ESI-MS approaches exhibit limitations regarding sensitivity and separation performance. Here, we report a new generic methodology for CIEF–ESI-MS based on chemical mobilization, leading to highly efficient separation. This new integrated methodology relies on exchanging catholyte, initially introduced in the nanoCEasy interface in the focusing step, with sheath liquid (SL) in order to chemically mobilize the analytes into the ESI-MS system. The CIEF–MS method is evaluated by separation of a peptide set, model proteins, and monoclonal antibody charge variants. The effect of various parameters including master mixture composition, field strength, catholyte, SL composition, focusing time, and capillary conditions is optimized and discussed. Excellent separation performance can be achieved with a pI resolution down to 0.1 pH unit. The mobilization reproducibility is demonstrated with "migration time" RSDs below 10%. Additionally, the chemical mobilization is compared with the pressure assistance–chemical mobilization method, demonstrating that even a small pressure causes a strong decrease in separation performance, which clearly indicates the benefit of the chemical mobilization-based method. The applicability and separation power of the developed method are further exhibited by separation of Fc-conjugated insulins (mass = 62 kDa) differing in only one amino acid.