Large-Scale Qualitative and Quantitative Top-Down Proteomics Using Capillary Zone Electrophoresis-Electrospray Ionization-Tandem Mass Spectrometry with Nanograms of Proteome Samples

Capillary zone electrophoresis-electrospray ionization-tandem mass spectrometry (CZE-ESI-MS/MS) has attracted attention recently for top-down proteomics because it can achieve highly efficient separation and very sensitive detection of proteins. However, separation window and sample loading volume of CZE need to be boosted for a better proteome coverage using CZE-MS/MS. Here, we present an improved CZE-MS/MS system that achieved a 180-min separation window and a 2-μL sample loading volume for top-down characterization of protein mixtures. The system obtained highly efficient separation of proteins with nearly one million theoretical plates for myoglobin and enabled baseline separation of three different proteoforms of myoglobin. The CZE-MS/MS system identified 797±21 proteoforms and 258±7 proteins (n=2) from an Escherichia coli (E. coli) proteome sample in a single run with only 250 ng of proteins injected. The system still identified 449±40 proteoforms and 173±6 proteins (n=2) from the E. coli sample when only 25 ng of proteins were injected per run. Single-shot CZE-MS/MS analyses of zebrafish brain cerebellum (Cb) and optic tectum (Teo) regions identified 1 730±196 proteoforms (n=3) and 2 024±255 proteoforms (n=3), respectively, with only 500-ng proteins loaded per run. Label-free quantitative top-down proteomics of zebrafish brain Cb and Teo regions revealed significant differences between Cb and Teo regarding the proteoform abundance. Over 700 proteoforms from 131 proteins had significantly higher abundance in Cb compared to Teo, and these proteins were highly enriched in several biological processes, including muscle contraction, glycolytic process, and mesenchyme migration

Capillary zone electrophoresis-tandem mass spectrometry with activated ion electron transfer dissociation for large-scale top-down proteomics

Capillary zone electrophoresis (CZE)-tandem mass spectrometry (MS/MS) has been recognized as an efficient approach for top-down proteomics recently for its high-capacity separation and highly sensitive detection of proteoforms. However, the commonly used collision-based dissociation methods often cannot provide extensive fragmentation of proteoforms for thorough characterization. Activated ion electron transfer dissociation (AI-ETD), that combines infrared photoactivation concurrent with ETD, has shown better performance for proteoform fragmentation than higher energy-collisional dissociation (HCD) and standard ETD. Here, we present the first application of CZE-AI-ETD on an Orbitrap Fusion Lumos mass spectrometer for large-scale top-down proteomics of Escherichia coli (E. coli) cells. CZE-AI-ETD outperformed CZE-ETD regarding proteoform and protein identifications (IDs). CZE-AI-ETD reached comparable proteoform and protein IDs with CZE-HCD. CZE-AI-ETD tended to generate better expectation values (E values) of proteoforms than CZE-HCD and CZE-ETD, indicating a higher quality of MS/MS spectra from AI-ETD respecting the number of sequence-informative fragment ions generated. CZE-AI-ETD showed great reproducibility regarding the proteoform and protein IDs with relative standard deviations less than 4% and 2% (n = 3). Coupling size exclusion chromatography (SEC) to CZE-AI-ETD identified 3028 proteoforms and 387 proteins from E. coli cells with 1% spectrum level and 5% proteoform-level false discovery rates. The data represents the largest top-down proteomics dataset using the AI-ETD method so far. Single-shot CZE-AI-ETD of one SEC fraction identified 957 proteoforms and 253 proteins. N-terminal truncations, signal peptide cleavage, N-terminal methionine removal, and various post-translational modifications including protein N-terminal acetylation, methylation, S-thiolation, disulfide bonds, and lysine succinylation were detected