scholarly journals Profiling Dopamine-Induced Oxidized Proteoforms of β-Synuclein by Top-Down Mass Spectrometry

Antioxidants ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 893
Author(s):  
Arianna Luise ◽  
Elena De Cecco ◽  
Erika Ponzini ◽  
Martina Sollazzo ◽  
PierLuigi Mauri ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Electron Transfer Dissociation ◽  
Native Mass Spectrometry ◽  
Biochemical Properties ◽  
Intact Protein ◽  
Top Down ◽  
Systematic Assessment ◽  
Methionine Residues ◽  
Stress States ◽  
Oxidation Of Methionine

The formation of multiple proteoforms by post-translational modifications (PTMs) enables a single protein to acquire distinct functional roles in its biological context. Oxidation of methionine residues (Met) is a common PTM, involved in physiological (e.g., signaling) and pathological (e.g., oxidative stress) states. This PTM typically maps at multiple protein sites, generating a heterogeneous population of proteoforms with specific biophysical and biochemical properties. The identification and quantitation of the variety of oxidized proteoforms originated under a given condition is required to assess the exact molecular nature of the species responsible for the process under investigation. In this work, the binding and oxidation of human β-synuclein (BS) by dopamine (DA) has been explored. Native mass spectrometry (MS) has been employed to analyze the interaction of BS with DA. In a second step, top-down fragmentation of the intact protein from denaturing conditions has been performed to identify and quantify the distinct proteoforms generated by DA-induced oxidation. The analysis of isobaric proteoforms is approached by a combination of electron-transfer dissociation (ETD) at each extent of modification, quantitation of methionine-containing fragments and combinatorial analysis of the fragmentation products by multiple linear regression. This procedure represents a promising approach to systematic assessment of proteoforms variety and their relative abundance. The method can be adapted, in principle, to any protein containing any number of methionine residues, allowing for a full structural characterization of the protein oxidation states.

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 Fixed-Charge Trimethyl Pyrilium Modification for Enabling Enhanced Top-Down Mass Spectrometry Sequencing of Intact Protein Complexes

2018 ◽  
Vol 90 (4) ◽  
pp. 2756-2764 ◽  
Author(s):  
Daniel A. Polasky ◽  
Frederik Lermyte ◽  
Michael Nshanian ◽  
Frank Sobott ◽  
Phillip C. Andrews ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Protein Complexes ◽  
Fixed Charge ◽  
Intact Protein ◽  
Top Down ◽  
Top Down Mass Spectrometry

scholarly journals An integrated native mass spectrometry and top-down proteomics method that connects sequence to structure and function of macromolecular complexes

2018 ◽  
Vol 10 (2) ◽  
pp. 139-148 ◽  
Author(s):  
Huilin Li ◽  
Hong Hanh Nguyen ◽  
Rachel R. Ogorzalek Loo ◽  
Iain D. G. Campuzano ◽  
Joseph A. Loo
Keyword(s):  
Mass Spectrometry ◽  
Native Mass Spectrometry ◽  
Structure And Function ◽  
Top Down ◽  
Macromolecular Complexes ◽  
And Function

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
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