Comparing nanoflow reversed-phase liquid chromatography-tandem mass spectrometry and capillary zone electrophoresis-tandem mass spectrometry for top-down proteomics

Abstract

One of the major shortcomings in top-down proteomics is the lack of efficient separations for intact proteins that can be effectively coupled to mass spectrometry. Capillary zone electrophoresis (CZE) and nanoflow reversed-phase liquid chromatography (nanoRPLC) are two methods that can be coupled to mass spectrometry directly and have been recently advanced in terms of their ability to separate intact proteins in complex biological mixtures. In this work, for the first time, we compared the state-of-the-art nanoRPLC-MS/MS and CZE-MS/MS platforms for top-down characterization of a standard protein mixture and an Escherichia coli (E. coli) proteome sample. CZE-MS produced comparable signals of standard proteins to RPLC-MS with 10-times less sample consumption. Interestingly, the proteins in RPLC-MS tended to have higher charge states than in CZE-MS, most likely due to the high acetonitrile concentration in RPLC mobile phase, leading to the more extensive unfolding of proteins in RPLC compared to in CZE. CZE-MS/MS identified 159 proteins and 513 proteoforms using 1-μg E. coli proteins in a single run and outperformed RPLC-MS/MS using 1-μg E. coli proteins in terms of protein and proteoform identifications (159 vs. 105 proteins and 513 vs. 277 proteoforms). The RPLC-MS/MS using 8-μg E. coli proteins identified 245 proteins and 1004 proteoforms in a single run, and the data was much better than that from CZE-MS/MS (1-μg E. coli proteins) regarding the number of identifications because of the 8-times higher sample loading amount and significantly wider separation window of RPLC-MS/MS compared to CZE-MS/MS.