Top-Down Proteomics Using Sheath-Flow Capillary Electrophoresis Coupled to Mass Spectrometry

While liquid chromatography dominates in the separation stage of proteomics studies, capillary electrophoresis is a valuable alternative and provides an orthogonal separation mode. It has proved its usefulness in bottom-up proteomics by generating complementary peptide identities. In this thesis, I present the applications of CZE-ESI-MS/MS in top-down proteomics. Capillary zone electrophoresis lends good resolving power to proteoforms with minor sequence variations or PTMs, which often have similar hydrophobicities but different charges. Here I apply this system to characterize secretome from M. marinum, demonstrate improve sequence coverage with combined fragmentation of HCD and AI-ETD, separate the heavy chains and light chains from reduced mAbs, and finally characterize proteins and proteoforms from yeast with prefractionation by RPLC. This work demonstrates the ability of CZE separating proteins and the potential for CZE-ESI-MS/MS as a platform for characterizing complex biological samples in top-down proteomics.