Intact protein analysis using capillary zone electrophoresis-mass spectrometry in different capillaries

Abstract

In this work, the performance of bare fused silica (BFS) capillary for intact protein analysis was compared to that of different (dynamically coated polybrene (PB) and permanently coated linear polyacrylamide (LPA)) coated capillaries using capillary zone electrophoresis - mass spectrometry (CZE-MS). In cases where low pH (pH=1.8) was used in bare silica capillaries for the separation of 5 destinct proteins, good precision (0.56-0.78 RSD% and 1.7-6.5 RSD% for migration times and peak areas, respectively), minimal adsorption and separation efficiency (N= 27 000/m -322 000/m) similar to or even better than those obtained with the coated capillaries was achieved. The PB and the LPA capillaries demonstrated their slightly better resolving power in terms of separating the different forms/variants of the same protein (e.g., hemoglobin subunits). The examined CZE-MS methods has been optimized for the separation of human insulin and its 6 analogues, where 3 layer PB-coated (PB-DS-PB) capilaries have exhibited the highest number of theoritical plates, resolution and precision data. A broader application areas of CZE-MS have also been explored by the application of the new methods for the separation of desamido insulin forms, native proteins and monoclonal antibody.