scholarly journals Fully automated chip-based nanoelectrospray combined with electron transfer dissociation for high throughput top-down proteomics

2013 ◽  
Vol 11 (1) ◽  
pp. 25-34 ◽  
Author(s):  
Corina Flangea ◽  
Catalin Schiopu ◽  
Florina Capitan ◽  
Cristina Mosoarca ◽  
Marilena Manea ◽  
...  
Keyword(s):  
Electron Transfer ◽  
High Throughput ◽  
Protein Identification ◽  
Electron Transfer Dissociation ◽  
Enzymatic Digestion ◽  
Protein Sequencing ◽  
Intact Protein ◽  
Medium Size ◽  
Ionization Mass ◽  
Top Down

AbstractThe conventional protocol for protein identification by electrospray ionization mass spectrometry (MS) is based on enzymatic digestion which renders peptides to be analyzed by liquid chromatography-MS and collision-induced dissociation (CID) multistage MS, in the so-called bottom-up approach. Though this method has brought a significant progress to the field, many limitations, among which, the low throughput and impossibility to characterize in detail posttranslational modifications in terms of site(s) and structure, were reported. Therefore, the research is presently focused on the development of procedures for efficient top-down fragmentation of intact protein ions. In this context, we developed here an approach combining fully automated chip-based-nanoelectrospray ionisation (nanoESI), performed on a NanoMate robot, with electron transfer dissociation (ETD) for peptide and top-down protein sequencing and identification. This advanced analytical platform, integrating robotics, microfluidics technology, ETD and alternate ETD/CID, was tested and found ideally suitable for structural investigation of peptides and modified/functionalized peptides as well as for top-down analysis of medium size proteins by tandem MS experiments of significantly increased throughput and sensitivity. The obtained results indicate that NanoMate-ETD and ETD/CID may represent a viable alternative to the current MS strategies, with potential to develop into a method of routine use for high throughput top-down proteomics.

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

2020 ◽  
Author(s):  
Muhammad Zenaidee ◽  
Carter Lantz ◽  
Taylor Perkins ◽  
Janine Fu ◽  
Wonhyuek Jung ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Electron Transfer Dissociation ◽  
Electron Ionization ◽  
Protein Sequencing ◽  
Intact Protein ◽  
Top Down ◽  
C Terminus ◽  
N Terminus ◽  
Product Ions ◽  
Protein Fragmentation

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.

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

2020 ◽  
Author(s):  
Muhammad Zenaidee ◽  
Carter Lantz ◽  
Taylor Perkins ◽  
Janine Fu ◽  
Wonhyuek Jung ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Electron Transfer Dissociation ◽  
Electron Ionization ◽  
Protein Sequencing ◽  
Intact Protein ◽  
Top Down ◽  
C Terminus ◽  
N Terminus ◽  
Product Ions ◽  
Protein Fragmentation

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.

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

2020 ◽  
Author(s):  
Muhammad Zenaidee ◽  
Carter Lantz ◽  
Taylor Perkins ◽  
Janine Fu ◽  
Wonhyuek Jung ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Electron Transfer Dissociation ◽  
Electron Ionization ◽  
Protein Sequencing ◽  
Intact Protein ◽  
Top Down ◽  
C Terminus ◽  
N Terminus ◽  
Product Ions ◽  
Protein Fragmentation

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.

scholarly journals The Value of Activated Ion Electron Transfer Dissociation for High-Throughput Top-Down Characterization of Intact Proteins

2018 ◽  
Vol 90 (14) ◽  
pp. 8553-8560 ◽  
Author(s):  
Nicholas M. Riley ◽  
Jacek W. Sikora ◽  
Henrique S. Seckler ◽  
Joseph B. Greer ◽  
Ryan T. Fellers ◽  
...  
Keyword(s):  
Electron Transfer ◽  
High Throughput ◽  
Electron Transfer Dissociation ◽  
Top Down ◽  
Intact Proteins

Automated Proteomics ofE. colivia Top-Down Electron-Transfer Dissociation Mass Spectrometry

2008 ◽  
Vol 80 (5) ◽  
pp. 1459-1467 ◽  
Author(s):  
Maureen K. Bunger ◽  
Benjamin J. Cargile ◽  
Anne Ngunjiri ◽  
Jonathan L. Bundy ◽  
James L. Stephenson
Keyword(s):  
Mass Spectrometry ◽  
Electron Transfer ◽  
Electron Transfer Dissociation ◽  
Top Down

scholarly journals Top-Down Characterization of Proteins with Intact Disulfide Bonds Using Activated-Ion Electron Transfer Dissociation

2018 ◽  
Vol 90 (15) ◽  
pp. 8946-8953 ◽  
Author(s):  
Matthew J. P. Rush ◽  
Nicholas M. Riley ◽  
Michael S. Westphall ◽  
Joshua J. Coon
Keyword(s):  
Electron Transfer ◽  
Disulfide Bonds ◽  
Electron Transfer Dissociation ◽  
Top Down
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