Ion Activation Methods for Peptides and Proteins

Brodbelt, Jennifer S.

doi:10.1021/acs.analchem.5b04563

Cited by 182 publications

(214 citation statements)

References 258 publications

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“…Extensive sequence information with retention of labile modifications and non-covalent ligand-bound fragment ions has been observed 22, 24, 26 . Sequence coverage for native and denatured proteins by UVPD was found to be independent of the charge state of the precursor ions 12 . Non-covalently bound protein-protein product ions have also been detected, which is useful tertiary and quaternary structure information 22 .…”

Section: Introductionmentioning

confidence: 97%

Structural Characterization of Native Proteins and Protein Complexes by Electron Ionization Dissociation-Mass Spectrometry

Sheng

McGee

et al. 2017

Anal. Chem.

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Mass spectrometry (MS) has played an increasingly important role in the identification and structural and functional characterization of proteins. In particular, the use of tandem mass spectrometry has afforded one of the most versatile methods to acquire structural information for proteins and protein complexes. The unique nature of electron capture dissociation (ECD) for cleaving protein backbone bonds while preserving non-covalent interactions has made it especially suitable for the study of native protein structures. However, the intra- and inter-molecular interactions stabilized by hydrogen bonds and salt bridges can hinder the separation of fragments even with pre-activation, which has become particularly problematic for the study of large macromolecular proteins and protein complexes. Here, we describe the capabilities of another activation method, 30 eV electron ionization dissociation (EID), for the top-down MS characterization of native protein-ligand and protein-protein complexes. Rich structural information that cannot be delivered by ECD can be generated by EID. EID allowed for the comparison of the gas-phase and the solution-phase structural stability and unfolding process of human carbonic anhydrase I (HCA-I). In addition, the EID fragmentation patterns reflect the structural similarities and differences among apo-, Zn-, and Cu,Zn-superoxide dismutase (SOD1) dimers. In particular, the structural changes due to Cu-binding and a point mutation (G41D) were revealed by EID-MS. The performance of EID was also compared to that of 193 nm ultraviolet photodissociation (UVPD), which allowed us to explore their qualitative similarities and differences as potential valuable tools for the MS study of native proteins and protein complexes.

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Section: Introductionmentioning

confidence: 97%

Structural Characterization of Native Proteins and Protein Complexes by Electron Ionization Dissociation-Mass Spectrometry

Sheng

McGee

et al. 2017

Anal. Chem.

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“…The process requires the measurement of the intact mass of the precursor, which is then isolated in the gas phase and activated to break its interresidue bonds. Many methods for activation and fragmentation are available (reviewed in [15, 16]), but one of the most common is via energetic collisions with a neutral gas, which is termed collisionally-induced dissociation (CID). Variations of CID exist, with the two major types being (1) ion trap CID, which uses resonant excitation to excite ions and potentiate radial movement that slowly dissociates trapped ions after many collisions, or (2) beam-style CID, which is performed using higher-energy axial acceleration to rapidly activate and dissociate the ions.…”

Section: Introductionmentioning

confidence: 99%

Defining Gas-Phase Fragmentation Propensities of Intact Proteins During Native Top-Down Mass Spectrometry

Haverland

Skinner

Fellers

et al. 2017

J. Am. Soc. Mass Spectrom.

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Fragmentation of intact proteins in the gas phase is influenced by amino acid composition, the mass and charge of precursor ions, higher order structure, and the dissociation technique used. The likelihood of fragmentation occurring between a pair of residues is referred to as the fragmentation propensity and is calculated by dividing the total number of assigned fragmentation events by the total number of possible fragmentation events for each residue pair. Here, we describe general fragmentation propensities when performing top-down mass spectrometry (TDMS) using denaturing or native electrospray ionization. A total of 5,311 matched fragmentation sites were collected for 131 proteoforms that were analyzed over 165 experiments using native top-down mass spectrometry (nTDMS). These data were used to determine the fragmentation propensities for 399 residue pairs. In comparison to denatured top-down mass spectrometry (dTDMS), the fragmentation pathways occurring either N-terminal to proline or C-terminal to aspartic acid were even more enhanced in nTDMS as compared to other residues. More generally, 257/399 (64%) of the fragmentation propensities were significantly altered (p ≤0.05) when using nTDMS as compared to dTDMS and of these, 123 were altered by 2-fold or greater. The most notable enhancements of fragmentation propensities for TDMS in native vs. denatured mode occurred (1) C-terminal to aspartic acid, (2) between phenylalanine and tryptophan (F|W), and (3) between tryptophan and alanine (W|A). The fragmentation propensities presented here will be of high value in the development of tailored scoring systems used in nTDMS of both intact proteins and protein complexes.

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“…Advances in high throughput bottom-up proteomics approaches [1, 2] have been driven in part by improvements to established ion activation methods and developments of new ones [3, 4]. New activation techniques, such as electron-based [5–8] and photoactivation methods [9–11], have resulted in the production of different types and distributions of diagnostic fragment ions than ones generated by conventional collisional activation methods.…”

Section: Introductionmentioning

confidence: 99%

Statistical Examination of the a and a + 1 Fragment Ions from 193 nm Ultraviolet Photodissociation Reveals Local Hydrogen Bonding Interactions

Morrison

Rosenberg

Singleton

et al. 2016

J. Am. Soc. Mass Spectrom.

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Dissociation of proteins and peptides by 193 nm ultraviolet photodissociation (UVPD) has gained momentum in proteomic studies due to the diversity of backbone fragments that are produced and subsequently unrivaled sequence coverage obtained by the approach. The pathways that form the basis for the production of particular ion types are not completely understood. In this study, a statistical approach is used to probe hydrogen atom elimination from a+1 radical ions, and different extents of elimination are found to vary as a function of the identity of the C-terminal residue of the a product ions and the presence or absence of hydrogen bonds to the cleaved residue.

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Ion Activation Methods for Peptides and Proteins

Abstract: Graphical Abstract

Cited by 182 publications

References 258 publications

Structural Characterization of Native Proteins and Protein Complexes by Electron Ionization Dissociation-Mass Spectrometry

Structural Characterization of Native Proteins and Protein Complexes by Electron Ionization Dissociation-Mass Spectrometry

Defining Gas-Phase Fragmentation Propensities of Intact Proteins During Native Top-Down Mass Spectrometry

Statistical Examination of the a and a + 1 Fragment Ions from 193 nm Ultraviolet Photodissociation Reveals Local Hydrogen Bonding Interactions

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