Activated Ion Electron Transfer Dissociation for Improved Fragmentation of Intact Proteins

Riley, Nicholas M.; Westphall, Michael S.; Coon, Joshua J.

doi:10.1021/acs.analchem.5b00881

Cited by 75 publications

(129 citation statements)

References 58 publications

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“…Future work will focus on how high capacity ETD can benefit other hybrid dissociation techniques, e.g. , ultraviolet photo-dissociation (UVPD)-ETD methods [74] and activated ion ETD (AI-ETD) [75, 76], with an emphasis on how this improved approach to ETD can be employed in large-scale proteome characterizations. With the implementation of high capacity ETD, we present a straightforward strategy to improve tandem mass spectra of intact proteins; accordingly, this approach is implemented on the Orbitrap Fusion Lumos and maintains all of the benefits of conducting ion-ion reactions in the dual-cell quadrupole linear ion trap.…”

Section: Resultsmentioning

confidence: 99%

Enhanced Dissociation of Intact Proteins with High Capacity Electron Transfer Dissociation

Riley

Mullen

Weisbrod

et al. 2015

J. Am. Soc. Mass Spectrom.

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Electron transfer dissociation (ETD) is a valuable tool for protein sequence analysis, especially for the fragmentation of intact proteins. However, low product ion signal-to-noise often requires some degree of signal averaging to achieve high quality MS/MS spectra of intact proteins. Here we describe a new implementation of ETD on the newest generation of quadrupole-Orbitrap-linear ion trap Tribrid, the Orbitrap Fusion Lumos, for improved product ion signal-to-noise via ETD reactions on larger precursor populations. In this new high precursor capacity ETD implementation, precursor cations are accumulated in the center section of the high pressure cell in the dual pressure linear ion trap prior to charge-sign independent trapping, rather than precursor ion sequestration in only the back section as is done for standard ETD. This new scheme increases the charge capacity of the precursor accumulation event, enabling storage of approximately three fold more precursor charges. High capacity ETD boosts the number of matching fragments identified in a single MS/MS event, reducing the need for spectral averaging. These improvements in intra-scan dynamic range via reaction of larger precursor populations, which have been previously demonstrated through custom modified hardware, are now available on a commercial platform, offering considerable benefits for intact protein analysis and top down proteomics. In this work, we characterize the advantages of high precursor capacity ETD through studies with myoglobin and carbonic anhydrase.

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Section: Resultsmentioning

confidence: 99%

Enhanced Dissociation of Intact Proteins with High Capacity Electron Transfer Dissociation

Riley

Mullen

Weisbrod

et al. 2015

J. Am. Soc. Mass Spectrom.

Self Cite

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“…Specifically, low-energy infrared photons have been used to increase the efficiency of ETD, in a process called activated ion ETD, resulting in a higher S/N of product ions observed in a single scan for model proteins (63). Additionally, 193-nm UV photons were used to characterize ribosomal proteins in LC-MS/MS experiments on complex samples—a fragmentation technique called UV photodissociation (UVPD) (64).…”

Section: Paths Forward In Top-down Proteomicsmentioning

confidence: 99%

Progress in Top-Down Proteomics and the Analysis of Proteoforms

Toby

Fornelli

Kelleher

2016

Annual Rev. Anal. Chem.

479

456

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From a molecular perspective, enactors of function in biology are intact proteins that can be variably modified at the genetic, transcriptional, or post-translational level. Over the past 30 years, mass spectrometry (MS) has become a powerful method for the analysis of proteomes. Prevailing bottom-up proteomics operates at the level of the peptide, leading to issues with protein inference, connectivity, and incomplete sequence/modification information. Top-down proteomics (TDP), alternatively, applies MS at the proteoform level to analyze intact proteins with diverse sources of intramolecular complexity preserved during analysis. Fortunately, advances in prefractionation workflows, MS instrumentation, and dissociation methods for whole-protein ions have helped TDP emerge as an accessible and potentially disruptive modality with increasingly translational value. In this review, we discuss technical and conceptual advances in TDP, along with the growing power of proteoform-resolved measurements in clinical and translational research.

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“…However, significant phosphate neutral loss associated with the NSD method warrants prospective examination of MBP PHOS sites via alternative fragmentation methods such as electron capture/transfer dissociation (ECD or ETD). 40 …”

Section: Resultsmentioning

confidence: 99%

Continuous Elution Proteoform Identification of Myelin Basic Protein by Superficially Porous Reversed-Phase Liquid Chromatography and Fourier Transform Mass Spectrometry

et al. 2017

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Myelin basic protein (MBP) plays an important structural and functional role in the neuronal myelin sheath. Translated MBP exhibits extreme microheterogeneity with numerous alternative splice variants (ASVs) and post-translational modifications (PTMs) reportedly tied to central nervous system maturation, myelin stability, and the pathobiology of various de- and dys-myelinating disorders. Conventional bioanalytical tools cannot efficiently examine ASV and PTM events simultaneously, which limits understanding of the role of MBP microheterogeneity in human physiology and disease. To address this need, we report on a top-down proteomics pipeline that combines superficially porous reversed-phase liquid chromatography (SPLC), Fourier transform mass spectrometry (FTMS), data-independent acquisition (DIA) with nozzle-skimmer dissociation (NSD), and aligned data processing resources to rapidly characterize abundant MBP proteoforms within murine tissue. The three-tier proteoform identification and characterization workflow resolved four known MBP ASVs and hundreds of differentially modified states from a single 90 min SPLC-FTMS run on ~0.5 μg of material. This included 323 proteoforms for the 14.1 kDa ASV alone. We also identified two novel ASVs from an alternative transcriptional start site (ATSS) of the MBP gene as well as a never before characterized S-acylation event linking palmitic acid, oleic acid, and stearic acid at C78 of the 17.125 kDa ASV.

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Activated Ion Electron Transfer Dissociation for Improved Fragmentation of Intact Proteins

Cited by 75 publications

References 58 publications

Enhanced Dissociation of Intact Proteins with High Capacity Electron Transfer Dissociation

Enhanced Dissociation of Intact Proteins with High Capacity Electron Transfer Dissociation

Progress in Top-Down Proteomics and the Analysis of Proteoforms

Continuous Elution Proteoform Identification of Myelin Basic Protein by Superficially Porous Reversed-Phase Liquid Chromatography and Fourier Transform Mass Spectrometry

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