Coupling Capillary Zone Electrophoresis with Electron Transfer Dissociation and Activated Ion Electron Transfer Dissociation for Top-Down Proteomics

Zhao, Yimeng; Riley, Nicholas M.; Sun, Liangliang; Hebert, Alexander S.; Yan, Xiaojing; Westphall, Michael S.; Rush, Matthew J. P.; Zhu, Guijie; Champion, Matthew M.; Medie, Felix Mba; Champion, Patricia A.; Coon, Joshua J.; Dovic̀hi, Norman J.

doi:10.1021/acs.analchem.5b00883

Cited by 52 publications

(44 citation statements)

References 61 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.

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“…More recently, Li et al (31) optimized this technique for TDP of 30–80-kDa proteoforms (the middle mass range that has proven resistant to traditional chromatography) using Pseudomonas aeruginosa whole-cell lysate. Capillary zone electrophoresis was also recently implemented for the first time online with top-down tandem MS utilizing electron transfer dissociation (ETD) instead of collision-based fragmentation methods, as a complementary technique to improve protein characterization on an electrophoretic timescale (32). …”

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.

467

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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“…CZE-ESI-MS/MS has been employed to analyze single protein proteoforms 14–15 including pharmaceutical protein glycoform profiling, 14 and more recently, complex biological samples. 16–18 CZE can be interfaced with mass spectrometers via electrospray ionization (ESI). Our group developed an electrokinetically pumped sheath-flow nanospray CZE-ESI-MS interface that transfers the analyte from the separation capillary to a glass emitter filled with sheath liquid for nanospray.…”

Section: Introductionmentioning

confidence: 99%

“…19,20 Its stability and sensitivity has been greatly improved with our third generation interface 21 and has been effective for various bottom-up 22–28 and top down proteomics analyses. 16–18,29,30 For example, 58 proteoforms were identified by a Q Exactive mass spectrometer from the Mycobacterium marinum secretome in a single shot experiment. 17 Another study coupled CZE to an Orbitrap Elite mass spectrometer through a prototype sheathless CESI interface 31 to characterize the Pyrococcus furiosus proteome and identified 134 protein groups and 291 proteoforms from three SPE column fractions.…”

Section: Introductionmentioning

confidence: 99%

Coupling Capillary Zone Electrophoresis to a Q Exactive HF Mass Spectrometer for Top-down Proteomics: 580 Proteoform Identifications from Yeast

et al. 2016

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We used reversed-phase liquid chromatography to separate the yeast proteome into 23 fractions. These fractions were then analyzed using capillary zone electrophoresis (CZE) coupled to a Q-Exactive HF mass spectrometer using an electrokinetically pumped sheath flow interface. The parameters of the mass spectrometer were first optimized for top-down proteomics using a mixture of seven model proteins; we observed that intact protein mode with trapping pressure of 0.2 and normalized collision energy of 20% produced the highest intact protein signals and most protein identifications. Then, we applied the optimized parameters for analysis of the fractionated yeast proteome. 580 proteoforms and 180 protein groups were identified via database searching of the MS/MS spectra. This number of proteoform identifications is two times larger than previous CZE-MS/MS studies. An additional 3,243 protein species were detected based on the parent ion spectra. Post-translational modifications including N-terminal acetylation, signal peptide removal, and oxidation were identified.

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Coupling Capillary Zone Electrophoresis with Electron Transfer Dissociation and Activated Ion Electron Transfer Dissociation for Top-Down Proteomics

Cited by 52 publications

References 61 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

Coupling Capillary Zone Electrophoresis to a Q Exactive HF Mass Spectrometer for Top-down Proteomics: 580 Proteoform Identifications from Yeast

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