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

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 bac...

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Autores principales: Michaelevski, Izhak, Kirshenbaum, Noam, Sharon, Michal
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MyJove Corporation 2010
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3149990/
https://www.ncbi.nlm.nih.gov/pubmed/20729801
http://dx.doi.org/10.3791/1985
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author Michaelevski, Izhak
Kirshenbaum, Noam
Sharon, Michal
author_facet Michaelevski, Izhak
Kirshenbaum, Noam
Sharon, Michal
author_sort Michaelevski, Izhak
collection PubMed
description 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(1). The power of the IM-MS approach lies in its ability to define the subunit packing and overall shape of protein assemblies at micromolar concentrations, and near-physiological conditions(1). Several recent IM studies of both individual proteins(2,3) and non-covalent protein complexes(4-9), 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). 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.
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spelling pubmed-31499902011-08-15 T-wave Ion Mobility-mass Spectrometry: Basic Experimental Procedures for Protein Complex Analysis Michaelevski, Izhak Kirshenbaum, Noam Sharon, Michal J Vis Exp Cellular Biology 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(1). The power of the IM-MS approach lies in its ability to define the subunit packing and overall shape of protein assemblies at micromolar concentrations, and near-physiological conditions(1). Several recent IM studies of both individual proteins(2,3) and non-covalent protein complexes(4-9), 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). 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. MyJove Corporation 2010-07-31 /pmc/articles/PMC3149990/ /pubmed/20729801 http://dx.doi.org/10.3791/1985 Text en Copyright © 2010, Journal of Visualized Experiments http://creativecommons.org/licenses/by-nc-nd/3.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported License. To view a copy of this license, visithttp://creativecommons.org/licenses/by-nc-nd/3.0/
spellingShingle Cellular Biology
Michaelevski, Izhak
Kirshenbaum, Noam
Sharon, Michal
T-wave Ion Mobility-mass Spectrometry: Basic Experimental Procedures for Protein Complex Analysis
title T-wave Ion Mobility-mass Spectrometry: Basic Experimental Procedures for Protein Complex Analysis
title_full T-wave Ion Mobility-mass Spectrometry: Basic Experimental Procedures for Protein Complex Analysis
title_fullStr T-wave Ion Mobility-mass Spectrometry: Basic Experimental Procedures for Protein Complex Analysis
title_full_unstemmed T-wave Ion Mobility-mass Spectrometry: Basic Experimental Procedures for Protein Complex Analysis
title_short T-wave Ion Mobility-mass Spectrometry: Basic Experimental Procedures for Protein Complex Analysis
title_sort t-wave ion mobility-mass spectrometry: basic experimental procedures for protein complex analysis
topic Cellular Biology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3149990/
https://www.ncbi.nlm.nih.gov/pubmed/20729801
http://dx.doi.org/10.3791/1985
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