Separation and Collision Cross Section Measurements of Protein Complexes Afforded by a Modular Drift Tube Coupled to an Orbitrap Mass Spectrometer

Sipe, Sarah N.; Sanders, James D.; Reinecke, Tobias; Clowers, Brian H.; Brodbelt, Jennifer S.

doi:10.1021/acs.analchem.2c01653

Cited by 8 publications

(9 citation statements)

References 74 publications

(140 reference statements)

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“…As previously described, having too few or too many ions injected at once may cause artificially inflated decay rates which yield overestimated CCS values. , The C-trap gas pressure was set low (0.1 to 1.0 values, corresponding to 8 × 10 –11 to 1 × 10 –10 mbar measuring using the UHV gauge), which was optimized to provide measurable signal decay for each peak. Ion mobility CCSs were collected using a home-built atmospheric pressure drift tube as previously described. ,, This drift tube consists of a 10 cm drift region and 10 cm desolvation region, the latter of which causes lower charge states than those produced by direct electrospray ionization, thus requiring the need for supercharging (IM experiments) and charge reducing (Orbitrap CCS experiments) additives to obtain comparable charge states. Specific charge states were not isolated for the IM experiments.…”

Section: Methodsmentioning

confidence: 99%

“…Ion mobility CCSs were collected using a home-built atmospheric pressure drift tube as previously described. 49,51,52 This drift tube consists of a 10 cm drift region and 10 cm desolvation region, the latter of which causes lower charge states than those produced by direct electrospray ionization, thus requiring the need for supercharging (IM experiments) and charge reducing (Orbitrap CCS experiments) additives to obtain comparable charge states. Specific charge states were not isolated for the IM experiments.…”

Section: Instrumentationmentioning

confidence: 99%

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Expanding Orbitrap Collision Cross-Section Measurements to Native Protein Applications Through Kinetic Energy and Signal Decay Analysis

et al. 2023

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The measurement of collision cross sections (CCS, σ) offers supplemental information about sizes and conformations of ions beyond mass analysis alone. We have previously shown that CCSs can be determined directly from the time-domain transient decay of ions in an Orbitrap mass analyzer as ions oscillate around the central electrode and collide with neutral gas, thus removing them from the ion packet. Herein, we develop the modified hard collision model, thus deviating from the prior FT-MS hard sphere model, to determine CCSs as a function of center-of-mass collision energy in the Orbitrap analyzer. With this model, we aim to increase the upper mass limit of CCS measurement for native-like proteins, characterized by low charge states and presumed to be in more compact conformations. We also combine CCS measurements with collision induced unfolding and tandem mass spectrometry experiments to monitor protein unfolding and disassembly of protein complexes and measure CCSs of ejected monomers from protein complexes.

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Section: Methodsmentioning

confidence: 99%

Section: Instrumentationmentioning

confidence: 99%

Expanding Orbitrap Collision Cross-Section Measurements to Native Protein Applications Through Kinetic Energy and Signal Decay Analysis

et al. 2023

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“…Ion mobility CCSs were collected using a home-built atmospheric pressure drift tube as previously described. 44,47,48 This drift tube consists of a 10 cm drift region and 10 cm desolvation region, the latter of which causes lower charge states than produced by direct electrospray ionization, thus requiring the need for supercharging (IM experiments) and charge reducing (Orbitrap CCS experiments) additives to obtain comparable charge states. Specific charge states were not isolated for the IM experiments.…”

Section: Instrumentationmentioning

confidence: 99%

Expanding Orbitrap collision cross section measurements to native protein applications through kinetic energy and signal decay analysis

James

Sanders

Fort

et al. 2022

Preprint

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The measurement of collision cross sections (CCS) offers supplemental information about sizes and conformations of ions beyond mass analysis alone. We have previously shown that CCSs can be determined directly from the time-domain transient decay of ions in an Orbitrap mass analyzer as ions oscillate around the central electrode and collide with neutral gas, thus removing them from the ion packet. Herein, we develop the soft sphere collision model, thus deviating from prior FT-MS CCS hard sphere model, to determine CCSs as a function of center-of-mass collision energy in the Orbitrap analyzer. With this model, we aim to increase the upper mass limit of CCS measurement for native-like proteins, characterized by low charge states and presumed to be in more compact conformations. We also combine CCS measurements with collision inducing unfolding and MS/MS experiments to monitor protein unfolding and disassembly of protein complexes and measure CCSs of ejected monomers from protein complexes.

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“…Understanding the structural heterogeneity of proteins and protein complexes in the gas phase remains a challenge even with the advancement of sophisticated tandem mass spectrometry, − ion mobility methods, − and new computational programs such as Alpha-Fold. , Changes imparted through interaction of proteins with ligands or other proteins, addition of post-translational modifications, or adoption of different conformations increases the difficulty of discerning structures. − Drift tube ion mobility has gained popularity as a way to provide gas-phase separation of heterogeneous ion populations based on their size and shape while allowing the determination of ion-neutral collision cross section (CCS). , …”

Section: Introductionmentioning

confidence: 99%

“…Orbitrap mass spectrometers provide enhanced mass resolution and MS/MS functionality but operate at significantly slower scan speeds (hundreds of milliseconds) than ion mobility separations (tens of milliseconds), posing a challenge for integration of drift tubes with high-performance mass analysis. Because of this duty-cycle mismatch, DTIM-Orbitrap instruments typically employ two electrostatic ion gates at the entrance and exit of the drift tube, which can be operated in such a way as to allow only ions with a specified drift time to be transmitted to the mass spectrometer. ,,,,,− Fourier transform (FT) multiplexing can be used to improve the duty cycle of standard dual-gate drift tube IM-MS systems and generate full arrival time distributions (ATDs) with improved sensitivity and shorter acquisition times. ,,− Through systematic modulation of the front and back gates of the drift tube using a swept frequency square waveform, the ion drift times are encoded as oscillations within ion current on the mass spectrometer. , Extracted ion chromatograms (XICs) can be used to selectively map the ion current for each m / z of interest prior to FT to generate m/z- specific ATDs.…”

Section: Introductionmentioning

confidence: 99%

Improving Ion Mobility Mass Spectrometry of Proteins through Tristate Gating and Optimization of Multiplexing Parameters

Butalewicz

Sanders

Clowers

et al. 2022

J. Am. Soc. Mass Spectrom.

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Coupling drift tube ion mobility (IM) to Fourier transform mass spectrometry (FT-MS) affords the opportunity for gas-phase separation of ions based on size and conformation with high-resolution mass analysis. However, combining IM and FT-MS is challenging because ions exit the drift tube on a much faster time scale than the rate of mass analysis. Fourier transform (FT) and Hadamard transform multiplexing methods have been implemented to overcome the duty-cycle mismatch, offering new avenues for obtaining high-resolution, high-mass-accuracy analysis of mobility-selected ions. The gating methods used to integrate the drift tube with the FT mass analyzer discriminate against the transmission of large, low-mobility ions owing to the well-known gate depletion effect. Tristate gating strategies have been shown to increase ion transmission for drift tube IM-FT-MS systems through implementation of dual ion gating, controlling the quantity and timing of ions through the drift tube to reduce losses of slow-moving ions. Here we present an optimized set of multiplexing parameters for tristate gating ion mobility of several proteins on an Orbitrap mass spectrometer and further report parameters for increased ion transmission and mobility resolution as well as decreased experimental times from 15 min down to 30 s. On average, peak intensities in the arrival time distributions (ATDs) for ubiquitin increased 2.1× on average, while those of myoglobin increased by 1.5× with a resolving power increase on average of 11%.

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Separation and Collision Cross Section Measurements of Protein Complexes Afforded by a Modular Drift Tube Coupled to an Orbitrap Mass Spectrometer

Cited by 8 publications

References 74 publications

Expanding Orbitrap Collision Cross-Section Measurements to Native Protein Applications Through Kinetic Energy and Signal Decay Analysis

Expanding Orbitrap Collision Cross-Section Measurements to Native Protein Applications Through Kinetic Energy and Signal Decay Analysis

Expanding Orbitrap collision cross section measurements to native protein applications through kinetic energy and signal decay analysis

Improving Ion Mobility Mass Spectrometry of Proteins through Tristate Gating and Optimization of Multiplexing Parameters

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