Methodology for measuring conformation of solvent-disrupted protein subunits using T-WAVE ion mobility MS: An investigation into eukaryotic initiation factors

Leary, Julie A.; Schenauer, Matthew R.; Ştefănescu, Raluca; Andaya, Armann; Ruotolo, Brandon T.; Robinson, Carol V.; Thalassinos, Konstantinos; Scrivens, James H.; Sokabe, Masaaki; Hershey, John W.B.

doi:10.1016/j.jasms.2009.05.003

Cited by 53 publications

(73 citation statements)

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“…The simultaneous measurement of Ω values of both the intact complex and disassembly products generates structural restraints which are then used for calculating topological models of the protein complexes. The basic assumption underlying this methodology is that the generated subcomplexes retain their native-like confirmations, and indeed recent studies have demonstrated that the solution structure of the disassembly products is maintained and no major rearrangement in either solution or gas phases have occurred 4,6 .…”

Section: Discussionmentioning

confidence: 99%

“…Several recent IM studies of both individual proteins 2,3 and non-covalent protein complexes [4][5][6][7][8][9] , successfully demonstrated that protein quaternary structure is maintained in the gas phase, and highlighted the potential of this approach in the study of protein assemblies of unknown geometry. Here, we provide a detailed description of IMS-MS analysis of protein complexes using the Synapt (Quadrupole-Ion Mobility-Time-of-Flight) HDMS instrument (Waters Ltd; the only commercial IM-MS instrument currently available) 10 .…”

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confidence: 99%

“…When defining the cross sectional area of the unknown protein or protein complex, we recommend that each experiment be repeated at least three times, and the standard deviation of these triplicate measurements determined. 4. Choose Display |2D Map Intensity Scale, and three options will appear: Linear Scale, Log Scale and Square Root Scale.…”

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T-wave Ion Mobility-mass Spectrometry: Basic Experimental Procedures for Protein Complex Analysis

Michaelevski

Kirshenbaum

Sharon

2010

JoVE

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Ion mobility (IM) is a method that measures the time taken for an ion to travel through a pressurized cell under the influence of a weak electric field. The speed by which the ions traverse the drift region depends on their size: large ions will experience a greater number of collisions with the background inert gas (usually N 2 ) and thus travel more slowly through the IM device than those ions that comprise a smaller cross-section. In general, the time it takes for the ions to migrate though the dense gas phase separates them, according to their collision cross-section (Ω).Recently, IM spectrometry was coupled with mass spectrometry and a traveling-wave (T-wave) Synapt ion mobility mass spectrometer (IM-MS) was released. Integrating mass spectrometry with ion mobility enables an extra dimension of sample separation and definition, yielding a three-dimensional spectrum (mass to charge, intensity, and drift time). This separation technique allows the spectral overlap to decrease, and enables resolution of heterogeneous complexes with very similar mass, or mass-to-charge ratios, but different drift times. Moreover, the drift time measurements provide an important layer of structural information, as Ω is related to the overall shape and topology of the ion. The correlation between the measured drift time values and Ω is calculated using a calibration curve generated from calibrant proteins with defined cross-sections , successfully demonstrated that protein quaternary structure is maintained in the gas phase, and highlighted the potential of this approach in the study of protein assemblies of unknown geometry. Here, we provide a detailed description of IMS-MS analysis of protein complexes using the Synapt (Quadrupole-Ion Mobility-Time-of-Flight) HDMS instrument (Waters Ltd; the only commercial IM-MS instrument currently available) 10 . We describe the basic optimization steps, the calibration of collision cross-sections, and methods for data processing and interpretation. The final step of the protocol discusses methods for calculating theoretical Ω values. Overall, the protocol does not attempt to cover every aspect of IM-MS characterization of protein assemblies; rather, its goal is to introduce the practical aspects of the method to new researchers in the field. Video LinkThe video component of this article can be found at https://www.jove.com/video/1985/ ProtocolThe procedure we describe focuses solely on IM-MS analysis of protein complexes. Therefore, we suggest that researchers unacquainted with the field of structural MS refer to the sample preparation steps, instrument calibration and MS and tandem MS optimization procedures described in Kirshenbaum et al. 2009 http://www.jove.com/index/details.stp?ID=1954. In general, this protocol involves low micromolar concentrations of complex (1-20 μM) in a volatile buffer such as ammonium acetate (0.005 -1 M, pH 6-8). Given that 1-2 μl are consumed per nanoflow capillary, we suggest 10-20 μl as a minimum volume, to enable optimization of MS conditions. Part...

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Section: Discussionmentioning

confidence: 99%

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T-wave Ion Mobility-mass Spectrometry: Basic Experimental Procedures for Protein Complex Analysis

Michaelevski

Kirshenbaum

Sharon

2010

JoVE

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“…In most cases, protein ions display a distribution of charge states when produced through nESI, and IM and MS measurements indicate that ions having the lowest ionic charge are the most "native-like" [11,13,24], in that IM data for ions of low charge states are, in general, the most-correlated to X-ray and nuclear magnetic resonance (NMR) datasets [25][26][27] and are often the most useful data points when constraining topological models of multiprotein complexes from IM-MS data [16]. For example, in early experiments involving tryptophan-RNA binding attenuation protein (TRAP) 11-mer ions, higher charge states were observed to be more compact relative to ions having lower charge [13].…”

Section: Introductionmentioning

confidence: 99%

Ion Mobility-Mass Spectrometry Reveals Conformational Changes in Charge Reduced Multiprotein Complexes

Bornschein

Hyung

Ruotolo

2011

J. Am. Soc. Mass Spectrom.

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Characterizing intact multiprotein complexes in terms of both their mass and size by ion mobilitymass spectrometry is becoming an increasingly important tool for structural biology. Furthermore, the charge states of intact protein complexes can dramatically influence the information content of gas-phase measurements performed. Specifically, protein complex charge state has a demonstrated influence upon the conformation, mass resolution, ion mobility resolution, and dissociation properties of protein assemblies upon collisional activation. Here we present the first comparison of charge-reduced multiprotein complexes generated by solution additives and gas-phase ion-neutral reaction chemistry. While the charge reduction mechanism for both methods is undoubtedly similar, significant gas-phase activation of the complex is required to reduce the charge of the assemblies generated using the solution additive strategy employed here. This activation step can act to unfold intact protein complexes, making the data difficult to correlate with solution-phase structures and topologies. We use ion mobility-mass spectrometry to chart such conformational effects for a range of multi-protein complexes, and demonstrate that approaches to reduce charge based on ion-neutral reaction chemistry in the gas-phase consistently produce protein assemblies having compact, 'native-like' geometries while the same molecules added in solution generate significantly unfolded gas-phase complexes having identical charge states.

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“…For example, solution-phase disruption approaches for producing low charge state protein monomers for IM-MS measurements is currently the dominant approach to the problem; however, such data collection requires extensive screening of solution conditions to remove subunit proteins from the assembly without significant remodeling or unfolding [9,12,[18][19][20][21][22][23]. An alternative method for generating IM information on compact protein subunits is through gas-phase dissociation.…”

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Ion Mobility-Mass Spectrometry Reveals Highly-Compact Intermediates in the Collision Induced Dissociation of Charge-Reduced Protein Complexes

Bornschein

Niu

Eschweiler

et al. 2015

J. Am. Soc. Mass Spectrom.

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Abstract. Protocols that aim to construct complete models of multiprotein complexes based on ion mobility and mass spectrometry data are becoming an important element of integrative structural biology efforts. However, the usefulness of such data is predicated, in part, on an ability to measure individual subunits removed from the complex while maintaining a compact/folded state. Gas-phase dissociation of intact complexes using collision induced dissociation is a potentially promising pathway for acquiring such protein monomer size information, but most product ions produced are possessed of high charge states and elongated/string-like conformations that are not useful in protein complex modeling. It has previously been demonstrated that the collision induced dissociation of charge-reduced protein complexes can produce compact subunit product ions; however, their formation mechanism is not well understood. Here, we present new experimental evidence for the avidin (64 kDa) and aldolase (157 kDa) tetramers that demonstrates significant complex remodeling during the dissociation of charge-reduced assemblies. Detailed analysis and modeling indicates that highly compact intermediates are accessed during the dissociation process by both complexes. Here, we present putative pathways that describe the formation of such ions, as well as discuss the broader significance of such data for structural biology applications moving forward.

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Methodology for measuring conformation of solvent-disrupted protein subunits using T-WAVE ion mobility MS: An investigation into eukaryotic initiation factors

Cited by 53 publications

References 35 publications

T-wave Ion Mobility-mass Spectrometry: Basic Experimental Procedures for Protein Complex Analysis

T-wave Ion Mobility-mass Spectrometry: Basic Experimental Procedures for Protein Complex Analysis

Ion Mobility-Mass Spectrometry Reveals Conformational Changes in Charge Reduced Multiprotein Complexes

Ion Mobility-Mass Spectrometry Reveals Highly-Compact Intermediates in the Collision Induced Dissociation of Charge-Reduced Protein Complexes

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