Single-shot top-down proteomics with capillary zone electrophoresis-electrospray ionization-tandem mass spectrometry for identification of nearly 600 Escherichia coli proteoforms

Capillary zone electrophoresis-electrospray ionization-tandem mass spectrometry (CZE-ESI-MS/MS) has been recognized as an invaluable platform for top-down proteomics. However, the scale of top-down proteomics from CZE-MS/MS is still limited due to the low loading capacity and narrow separation window of CZE. In this work, for the first time we systematically evaluated dynamic pH junction method for focusing of intact proteins during CZE-MS. The optimized dynamic pH junction based CZE-MS/MS system approached 1-µL loading capacity, 90-min separation window and high peak capacity (~280) for separation of Escherichia coli proteome. The results represent the largest loading capacity and the highest peak capacity of CZE for top-down characterization of complex proteomes. About 2,800 proteoform-spectrum matches, nearly 600 proteoforms, and 200 proteins were identified from an Escherichia coli lysate by single-shot CZE-MS/MS with spectrum-level false discovery rate (FDR) less than 1%. The number of proteoforms is over three times higher than that from previous single-shot CZE-MS/MS.