Capillary Zone Electrophoresis-Tandem Mass Spectrometry for Top-Down Proteomics of Mouse Brain Integral Membrane Proteins

Massspectrometry (MS)-based top-down characterization of integralmembrane proteins (IMPs) is crucial for understanding their functionsin biological processes. However, it is technically challenging dueto their low solubility in typical MS-compatible buffers. In thiswork, for the first time, we developed an efficient capillary zoneelectrophoresis (CZE)-tandem MS (MS/MS) method for the top-down proteomics(TDP) of IMPs enriched from mouse brains. Our technique employs asample buffer containing 30% (v/v) formic acid and 60% (v/v) methanolfor solubilizing IMPs and utilizes a separation buffer of 30% (v/v)acetic acid and 30% (v/v) methanol for maintaining the solubilityof IMPs during CZE separation. Single-shot CZE-MS/MS identified 51IMP proteoforms from the mouse brain sample. Coupling size exclusionchromatography (SEC) to CZE-MS/MS enabled the identification of 276IMP proteoforms from the mouse brain sample containing 1-4transmembrane domains. This proof-of-concept work demonstrates thehigh potential of CZE-MS/MS for the large-scale TDP of IMPs.