It is critically important to study protein function to elucidate key biological processes such as molecular mechanisms of diseases. Structural changes in proteins (e.g., posttranslational modifications (PTMs) and truncations) give rise to numerous biologically relevant proteoforms that can impact/influence protein assembly, conformation, and function, thereby affecting cellular activity. However, protein structural changes have been challenging to investigate via conventional bottom-up proteomics, so there is growing interest in proteomics-based methods that focus on intact protein analysis (e.g., top-down proteomics (TDP) and native mass spectrometry (native MS)). Although significant progress has been made in the field of intact protein analysis by MS over the past decades, it is still notoriously challenging to achieve high-separation efficiency and extensive fragmentation of proteoforms and protein assemblies. As a result, interest in analytical tools with high separation efficiency such as capillary electrophoresis (CE) and in fragmentation techniques with high dissociation power such as electron-transfer/higher-energy collision dissociation (EThcD) and ultraviolet photodissociation (UVPD) has increased. We have recently shown that combining datasets from the analyses of a bovine seminal plasma using capillary zone electrophoresis (CZE) and reversed phase liquid-chromatography (LC) increases the number of proteoform identifications, due to the different separation mechanisms of CZE and LC. We have also demonstrated that either EThcD or 213 nm UVPD can generate satisfactory fragmentation of intact protein ions. EThcD returned better scoring metrics and sequence coverage, but 213 nm UVPD provided a higher number of identifications. In my talk, I will trace the history of our previous work and highlight our current efforts which have focused on the use of CE as a direct infusion platform for analysis of non-covalent protein complexes. The performance of CE as a direct infusion platform is further evaluated by integrating it with trapped ion mobility.

The versatility of capillary electrophoresis-mass spectrometry to intact protein analysis

ACS Fall 2021

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