Capillary zone electrophoresis-tandem mass spectrometry for top-down proteomics of mouse brain integral membrane proteins

ABSTRACT: Mass spectrometry (MS)-based top-down characterization of integral membrane proteins (IMPs) is crucial for understanding their functions in biological processes. However, it is technically challenging due to their low solubility in typical MS-compatible buffers. In this work, for the first time, we developed an efficient capillary zone electrophoresis (CZE)-tandem MS (MS/MS) method for top-down proteomics (TDP) of IMPs enriched from mouse brains. Our technique employs a sample buffer containing 30% (v/v) formic acid and 60% (v/v) methanol for solubilizing IMPs and utilizes a separation buffer of 30% (v/v) acetic acid and 30% (v/v) methanol for maintaining the solubility of IMPs during CZE separation. Single-shot CZE-MS/MS identified 51 IMP proteoforms from the mouse brain sample. Coupling size exclusion chromatography (SEC) to CZE-MS/MS enabled the identification of 276 IMP proteoforms from the mouse brain sample and those proteoforms contained 1-4 transmembrane domains. This proof-of-concept work demonstrates the high potential of CZE-MS/MS for large-scale TDP of IMPs.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Brain

SUBMITTER: Liangliang Sun  

LAB HEAD: Liangliang Sun

PROVIDER: PXD042298 | Pride | 2023-12-07

REPOSITORIES: Pride