| Autor: |
McCool EN; Department of Chemistry, Michigan State University, 578 S Shaw Lane, East Lansing, MI, 48824, USA., Lodge JM; Genome Center of Wisconsin, University of Wisconsin-Madison, Madison, WI, 53706, USA.; Department of Chemistry, University of Wisconsin-Madison, Madison, WI, 53706, USA., Basharat AR; Department of BioHealth Informatics, Indiana University-Purdue University Indianapolis, 719 Avenue, Indianapolis, IN, 46202, USA., Liu X; Department of BioHealth Informatics, Indiana University-Purdue University Indianapolis, 719 Avenue, Indianapolis, IN, 46202, USA.; Center for Computational Biology and Bioinformatics, Indiana University School of Medicine, 410 West 10th Street, Indianapolis, IN, 46202, USA., Coon JJ; Genome Center of Wisconsin, University of Wisconsin-Madison, Madison, WI, 53706, USA.; Department of Chemistry, University of Wisconsin-Madison, Madison, WI, 53706, USA.; Department of Biomolecular Chemistry, University of Wisconsin-Madison, Madison, WI, 53706, USA., Sun L; Department of Chemistry, Michigan State University, 578 S Shaw Lane, East Lansing, MI, 48824, USA. lsun@chemistry.msu.edu. |
| Abstrakt: |
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. |
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