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

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.

Download Full-text

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

10.26434/chemrxiv.11573337 ◽

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.

Download Full-text

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

Antioxidants ◽

10.3390/antiox10060893 ◽

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.

Download Full-text

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

Open Chemistry ◽

10.2478/s11532-012-0130-2 ◽

2013 ◽

Vol 11 (1) ◽

pp. 25-34 ◽

Cited By ~ 7

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.

Download Full-text

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

Analytical Chemistry ◽

10.1021/acs.analchem.7b04806 ◽

2018 ◽

Vol 90 (4) ◽

pp. 2756-2764 ◽

Cited By ~ 9

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

Download Full-text

Fast Top-Down Intact Protein Characterization with Capillary Zone Electrophoresis–Electrospray Ionization Tandem Mass Spectrometry

Analytical Chemistry ◽

10.1021/ac4008122 ◽

2013 ◽

Vol 85 (12) ◽

pp. 5989-5995 ◽

Cited By ~ 47

Author(s):

Liangliang Sun ◽

Michael D. Knierman ◽

Guijie Zhu ◽

Norman J. Dovichi

Keyword(s):

Mass Spectrometry ◽

Tandem Mass Spectrometry ◽

Electrospray Ionization ◽

Protein Characterization ◽

Intact Protein ◽

Tandem Mass ◽

Top Down ◽

Zone Electrophoresis ◽

Capillary Zone ◽

Ionization Tandem Mass Spectrometry

Download Full-text

Escherichia coli l-aspartate-α-decarboxylase: preprotein processing and observation of reaction intermediates by electrospray mass spectrometry

Biochemical Journal ◽

10.1042/bj3230661 ◽

1997 ◽

Vol 323 (3) ◽

pp. 661-669 ◽

Cited By ~ 53

Author(s):

Manoj K. RAMJEE ◽

Ulrich GENSCHEL ◽

Chris ABELL ◽

Alison G. SMITH

Keyword(s):

Mass Spectrometry ◽

Escherichia Coli ◽

Electrospray Mass Spectrometry ◽

Elevated Temperatures ◽

Gene Encoding ◽

Α Subunit ◽

C Terminus ◽

N Terminus ◽

Terminal Amino ◽

Α Subunits

The Escherichia coli panD gene, encoding l-aspartate-α-decarboxylase, was cloned by PCR, and shown to complement apanD mutant defective in β-alanine biosynthesis. Aspartate decarboxylase is a pyruvoyl-dependent enzyme, and is synthesized initially as an inactive proenzyme (the π-protein), which is proteolytically cleaved at a specific X–Ser bond to produce a β-subunit with XOH at its C-terminus and an α-subunit with a pyruvoyl group at its N-terminus, derived from the serine. The recombinant enzyme, as purified, is a tetramer, and comprises principally the unprocessed π-subunit (of 13.8 kDa), with a small proportion of the α- and β-subunits (11 kDa and 2.8 kDa respectively). Incubation of the purified enzyme at elevated temperatures for several hours results in further processing. Using fluorescein thiosemicarbazide, the completely processed enzyme was shown to contain three pyruvoyl groups per tetrameric enzyme. The presence of unchanged serine at the N-terminus of some of the α-subunits was confirmed by electrospray mass spectrometry (ESMS) and N-terminal amino acid sequencing. A novel HPLC assay for aspartate decarboxylase was established and used to determine the Km and kcat for l-aspartate as 151±16 μM and 0.57 s-1 respectively. ESMS was also used to observe substrate and product adducts trapped on the pyruvoyl group by sodium cyanoborohydride treatment.

Download Full-text

A Novel Thrombin Fluorogenic Substrate of High Affinity, Catalytic Efficiency and Selectivity.

Blood ◽

10.1182/blood.v106.11.1953.1953 ◽

2005 ◽

Vol 106 (11) ◽

pp. 1953-1953

Author(s):

T. Regan Baird ◽

Margaret Jacobs ◽

Adam Tinklepaugh ◽

Peter Gross ◽

Barbara C. Furie ◽

...

Keyword(s):

Spectral Properties ◽

Protein Sequencing ◽

Kinetic Properties ◽

Fluorogenic Substrate ◽

High Affinity ◽

Thrombin Activity ◽

Terminal Fragment ◽

C Terminus ◽

N Terminus ◽

Hydrolysis Of

Abstract Thrombin is a serine protease with multiple functions, including the conversion of fibrinogen to fibrin, platelet activation, activation of Factor VIII and Factor V. Although many low molecular weight substrates have been developed for the study of thrombin catalytic activity, our interest in analyzing thrombin activity in the blood of a living mouse required development of a new class of thrombin substrates of high affinity and high selectivity whose product upon hydrolysis could be visualized by intravital fluorescence microscopy. We have developed a novel substrate for thrombin using the fluorochrome Alexa 488 and the quencher QSY35. Alexa 488 is conjugated to the N-terminus of a 12 amino acid peptide based upon the thrombin cleavage site in the α-chain of fibrinogen and the quencher is coupled to the C-terminus of the peptide, yielding Alexa 488-KGGVR-GPRVVEA-QSY35. We term this substrate FBG-12. Through fluorescence energy resonance transfer, the emission from Alexa 488 is absorbed by the quencher QSY 35, thus minimizing fluorescence, since the two moieties have overlapping spectral properties and are separated by a Förster radius less than 44 Å. Thrombin hydrolyzes the peptide yielding the N-terminal fragment, Alexa 488-KGGVR-COOH, which is highly fluorescent, and the C-terminal fragment NH2-GPRVVEA-QSY35 peptide which, being physically separated from the Alexa 488, no longer quenches the fluorochrome. The peptide was synthesized by solid phase peptide synthesis, its N-terminus and C-terminus were modified with Alexa 488 and QSY35 respectively, and its identity confirmed by mass spectroscopy and protein sequencing. The kinetic properties of FBG-12 were determined in vitro. Hydrolysis of the substrate by thrombin resulted in a linear increase in fluorescence at 525 nm over time and was dependent on enzyme concentration. The fluorescence of the product of thrombin hydrolysis of FBG-12 was 120-fold greater than that of the substrate. Michaelis-Menten kinetic analysis of thrombin hydrolysis of the fluorogenic substrate FBG-12 revealed a Km of 2 μM, a kcat of 759 s−1, and a kcat/Km of 3795 x 106 M−1 s−1. To determine the selectivity of this substrate, other plasma serine proteases were analyzed for their ability to hydrolyze FBG-12. Factor Xa, Factor VIIa, and activated protein C did not hydrolyze FBG-12. Factor XIa (Km 11 μM, kcat 20s−1, kcat/Km = 20 x 106 M−1 s−1) did hydrolyze FBG-12, although the reaction was inefficient compared to thrombin. Our characterization of FBG-12 suggests that this substrate is hydrolyzed efficiently and selectively by thrombin. Its spectral properties and solubility in a physiologic environment make FBG-12 a suitable substrate for the detection of thrombin activity via intravital imaging during thrombus formation in vivo.

Download Full-text