Activated-Ion Electron Transfer Dissociation Improves the Ability of Electron Transfer Dissociation to Identify Peptides in a Complex Mixture

Ledvina, Aaron R.; Beauchene, Nicole A.; McAlister, Graeme C.; Syka, John E. P.; Schwartz, Jae C.; Griep-Raming, Jens; Westphall, Michael S.; Coon, Joshua J.

doi:10.1021/ac1020358

Cited by 69 publications

(82 citation statements)

References 45 publications

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“…The 200 ms spectra are reported. IRMPD and energy-resolved CID experiments were carried out on a modified dual cell QLT outfitted with a chemical ionization source for the generation of fluoranthene anion radicals, as described previously [23]. The mass spectrometer was modified to allow for the introduction of IR photons to the ion trapping region of the QLT.…”

Section: Methodsmentioning

confidence: 99%

Competitive hydrogen atom migrations accompanying cascade dissociations of peptide cation-radicals of the + type

Ledvina

Coon

Tureček

2015

International Journal of Mass Spectrometry

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We report a combined experimental and computational study of energy-resolved collision-induced dissociation (ER-CID) and time-resolved infrared multiphoton dissociation (TR-IRMPD) of z4 ions prepared by electron transfer dissociation of peptide (Ala-Ala-Asn-Ala-Arg + 2H)2+ ions. The z4 cation-radicals, •ANAR+, undergo competitive dissociations by backbone cleavage and loss of a CONH2 radical from the Asn side chain. The backbone cleavage proceeds by radical-assisted dissociation of the Asn Cα—CO bond, forming an x2 ion intermediate which rapidly dissociates by HNCO elimination to yield a stable z2 fragment ion, •AR+. The ER-CID and TR-IRMPD data were consistent with the consecutive nature of the backbone dissociation but showed different branching ratios for the two major fragmentations. The ER-CID data showed branching ratios 0.6-1.0 for the side-chain and backbone cleavages whereas the TR-IRMPD data showed an earlier onset for the latter dissociation. Computational analysis of the potential energy surface with density functional theory and ab initio calculations was carried out to provide structures and energies for the reactant ions as well as several intermediates, products, and transition states. Dissociation pathways for cis and trans amide conformers were distinguished and their energies were evaluated. The threshold dissociation energies for the backbone and side-chain dissociations were similar in accordance with the experimental ER-CID branching ratio. The TR-IRMPD data were interpreted by different absorbances of intermediates produced by hydrogen atom migrations along the dissociation pathways.

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Section: Methodsmentioning

confidence: 99%

Competitive hydrogen atom migrations accompanying cascade dissociations of peptide cation-radicals of the + type

Ledvina

Coon

Tureček

2015

International Journal of Mass Spectrometry

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“…A further increase in SNR obtained by adding more LC-MS/MS runs is feasible, although the envisioned SNR gain might not be justified by the increased sample consumption and experiment duration. Therefore, other ways of improving SNR in a single LC-MS/MS experiment are to be explored, including (i) activated ion-ETD (35,41), (ii) protein ion supercharging (42)(43)(44)(45), (iii) an increased number of precursor ions obtained through multiple fills of the ETD reaction cell, (iv) improved product ion transmission to the Orbitrap mass analyzer from the ETD reaction cell, and (v) an increased number of scans acquired per LC-MS/MS experiment via super-resolutionmethod-based signal processing applied complementary to the FT signal processing (46).…”

Section: Top-down Etd Orbitrap Ftms Of Igg1mentioning

confidence: 99%

Analysis of Intact Monoclonal Antibody IgG1 by Electron Transfer Dissociation Orbitrap FTMS

Fornelli

Damoc

Thomas

et al. 2012

Molecular & Cellular Proteomics

151

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The primary structural information of proteins employed as biotherapeutics is essential if one wishes to understand their structure-function relationship, as well as in the rational design of new therapeutics and for quality control. Given both the large size (around 150 kDa) and the structural complexity of intact immunoglobulin G (IgG), which includes a variable number of disulfide bridges, its extensive fragmentation and subsequent sequence determination by means of tandem mass spectrometry ( Top-down mass spectrometry (MS) 1 (1-3) has continued to demonstrate its particular advantages over traditionally employed bottom-up MS strategies (4). Specifically, top-down MS allows the characterization of specific protein isoforms originating from the alternative splicing of mRNA that code single nucleotide polymorphisms and/or post-translational modifications (PTMs) of protein species (5). Intact protein molecular weight (MW) determination and subsequent gasphase fragmentation of selected multiply charged protein ions (referred to as tandem MS or MS/MS) theoretically might result in complete protein sequence coverage and precise assignment of the type and position of PTMs, amino acid substitutions, and C-or N-terminal truncations (6), whereas the bottom-up MS approach allows only the identification of a certain protein family when few or redundant peptides are found for a particular protein isoform. At a practical level, however, top-down MS-based proteomics struggles not only with the single-or multi-dimensional separation of undigested proteins, which demonstrates lower reproducibility and repeatability than for peptides, but also with technical limitations present in even state-of-the-art mass spectrometers. The outcome of a top-down MS experiment depends indeed on the balance between the applied resolution of the mass spectrometer and its sensitivity. The former is required for unambiguous assignment of ion isotopic clusters in both survey and MS/MS scans, whereas the latter is ultimately dependent on the scan speed of the mass analyzer, which determines the number of scans that can be accumulated for a given analyte ion on the liquid chromatography (LC) timescale to enhance the resulting signal-to-noise ratio (SNR). Until recently, the instrument of choice for top-down MS has been the Fourier transform ion cyclotron resonance (FT-ICR) mass From the ‡Biomolecular Mass

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“…35−37 Instead, we have introduced a viable alternative, IR photon (10.6 μm) irradiation concurrent to the ETD reaction, termed activated ion ETD (AI-ETD), and have shown the benefits it affords peptide fragmentation for shotgun analyses. 30,38,39 Despite the success of AI-ETD in peptide fragmentation and the enhancements seen with AI-ECD for intact protein analysis, AI-ETD has yet to be widely applied to top down proteomics. Where ECD and AI-ECD are largely limited to FT-ICR instruments, AI-ETD can be implemented on virtually any instrument that has an rf trapping device, making it an appealing tool for ubiquitous use in the top down field.…”

mentioning

confidence: 99%

Activated Ion Electron Transfer Dissociation for Improved Fragmentation of Intact Proteins

2015

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Here we report the first implementation of activated ion electron transfer dissociation (AI-ETD) for top down protein characterization, showing that AI-ETD definitively extends the m/z range over which ETD can be effective for fragmentation of intact proteins. AI-ETD, which leverages infrared photon bombardment concurrent to the ETD reaction to mitigate nondissociative electron transfer, was performed using a novel multipurpose dissociation cell that can perform both beam-type collisional dissociation and ion-ion reactions on an ion trap-Orbitrap hybrid mass spectrometer. AI-ETD increased the number of c- and z-type product ions for all charge states over ETD alone, boosting product ion yield by nearly 4-fold for low charge density precursors. AI-ETD also outperformed HCD, generating more matching fragments for all proteins at all charge states investigated. In addition to generating more unique fragment ions, AI-ETD provided greater protein sequence coverage compared to both HCD and ETD. In all, the effectiveness of AI-ETD across the entirety of the m/z spectrum demonstrates its efficacy for robust fragmentation of intact proteins.

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Activated-Ion Electron Transfer Dissociation Improves the Ability of Electron Transfer Dissociation to Identify Peptides in a Complex Mixture

Cited by 69 publications

References 45 publications

Competitive hydrogen atom migrations accompanying cascade dissociations of peptide cation-radicals of the + type

Competitive hydrogen atom migrations accompanying cascade dissociations of peptide cation-radicals of the + type

Analysis of Intact Monoclonal Antibody IgG1 by Electron Transfer Dissociation Orbitrap FTMS

Activated Ion Electron Transfer Dissociation for Improved Fragmentation of Intact Proteins

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