Single-Shot Top-Down Proteomics with Capillary Zone Electrophoresis-Electrospray Ionization-Tandem Mass Spectrometry for Identification of Nearly 600 Escherichia coli Proteoforms

Abstract

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 using 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 the dynamic pH junction method for focusing of intact proteins during CZE-MS. The optimized dynamic pH junction based CZE-MS/MS approached 1-μL loading capacity, 90-min separation window and high peak capacity (~280) for characterization of an Escherichia coli proteome. The results represent the largest loading capacity and the highest peak capacity of CZE for top-down characterization of complex proteomes. Single-shot CZE-MS/MS identified about 2,800 proteoform-spectrum matches, nearly 600 proteoforms, and 200 proteins from the Escherichia coli proteome with spectrum-level false discovery rate (FDR) less than 1%. The number of identified proteoforms in this work is over three times higher than that in previous single-shot CZE-MS/MS studies. Truncations, N-terminal methionine excision, signal peptide removal and some post-translational modifications including oxidation and acetylation were detected.

Recent advances in capillary electrophoresis‐mass spectrometry: Instrumentation, methodology and applications

“…They were able to separate and identify truncated, oxidized and even deamidated forms. CZE‐MS was successfully applied for top‐down characterization of the E. coli proteome . By combining an optimized separation method with a dynamic pH junction stacking method, injection of up to 1 μL sample was possible and resulted in the identification of 600 proteoforms of 200 proteins.…”

Recent progress of sample stacking in capillary electrophoresis (2016–2018)

“…Lubeckyj et al. evaluated the dynamic pH junction method for focusing of intact proteins during CZE‐MS/MS for top‐down proteomics. Different BGEs were used (5–10% HAc or 0.1–0.5% formic acid), the sheath buffer was 0.2% formic acid containing 10% MeOH and the sample buffer contained 10 mM NH 4 HCO 3 at pH 8.0.…”

Identification and Quantification of Proteoforms by Mass Spectrometry

“…The Sun group recently made progress toward increasing the sample loading volume and the separation window of CZE. They achieved a 90‐min separation window and microliter‐scale sample loading volume for CZE‐MS/MS analysis of an E. coli cell lysate and identified 600 proteoforms in a single run . To facilitate the wide separation window and increased sample volume, a separation capillary with a high‐quality neutral coating on its inner wall and a highly efficient protein stacking method based on a dynamic pH junction principle were utilized.…”