ChemRxiv
These are preliminary reports that have not been peer-reviewed. They should not be regarded as conclusive, guide clinical practice/health-related behavior, or be reported in news media as established information. For more information, please see our FAQs.
1/1
2 files

Internal Fragments Generated by Electron Ionization Dissociation Enhances Protein Top-down Mass Spectrometry

preprint
revised on 26.02.2020 and posted on 27.02.2020 by Muhammad Zenaidee, Carter Lantz, Taylor Perkins, Janine Fu, Wonhyuek Jung, Rachel R. Ogorzalek Loo, Joseph A. Loo
Top-down proteomics by mass spectrometry (MS) involves the mass measurement of an intact protein followed by subsequent activation of the protein to generate product ions. Electron-based fragmentation methods like electron capture dissociation (ECD) and electron transfer dissociation (ETD) are widely used for these types of analysis, however these fragmentation methods can be inefficient due to the low energy electrons fragmenting the protein without the dissociation products; that is no detection of fragments formed. Recently, electron ionization dissociation (EID), which utilizes higher energy electrons (> 20 eV) has been shown to be more efficient for top-down protein fragmentation compared to other electron-based dissociation methods. Here we demonstrate that the use of EID enhances protein fragmentation and subsequent detection of protein fragments. Protein product ions can form by either single cleavage events, resulting in terminal fragments containing the C-terminus or N-terminus of the protein, or by multiple cleavage events to give rise to internal fragments that do not contain the C-terminus or N-terminus of the protein. Conventionally, internal fragments have been disregarded as reliable assignments of these fragments were limited. Here, we demonstrate that internal fragments generated by EID can account for ~20-40% of the mass spectral signals detected by top-down EID-MS experiments. By including internal fragments, the extent of the protein sequence that can be explained from a single tandem mass spectrum increases from ~50% to ~99% for 29 kDa carbonic anhydrase II and 8.6 kDa ubiquitin. By including internal fragments in the data analysis, previously unassigned peaks can be readily and accurately assigned to enhance the efficiencies of top-down protein sequencing experiments.

History

Email Address of Submitting Author

mzenaidee@ucla.edu

Institution

University of California, Los Angeles

Country

United States

ORCID For Submitting Author

0000-0003-3188-1472

Declaration of Conflict of Interest

No conflict of interest

Version Notes

Version 1.0

Exports

Read the published paper

in Journal of the American Society for Mass Spectrometry

Logo branding

Exports