Native IM-Orbitrap MS: Resolving what was hidden

Poltash, Michael L.; McCabe, Jacob W.; Shirzadeh, Mehdi; Laganowsky, Arthur; Russell, David H.

doi:10.1016/j.trac.2019.05.035

Cited by 39 publications

(58 citation statements)

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“…The observation of FXN cold denaturation by vT-IM-MS in this study highlights the capability of the new device to resolve subtle structural changes that were previously hidden via traditional biophysical techniques. 47 The activities of ion channels and chaperones are highly regulated by conformational ("open" and "closed") changes that are induced or stabilized by ligand binding that are subject to both enthalpic and entropic barriers. 20 GroEL (HSP60, 800 kDa), a 14-mer complex of the HSP10 monomers that are arranged as two stacked heptamers (see Figure 4), forms a large central cavity in which non-native proteins bind via hydrophobic interactions.…”

Section: ■ Results and Discussionmentioning

confidence: 99%

Variable-Temperature Electrospray Ionization for Temperature-Dependent Folding/Refolding Reactions of Proteins and Ligand Binding

et al. 2021

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Stabilities and structure(s) of proteins are directly coupled to their local environment or Gibbs free energy landscape as defined by solvent, temperature, pressure, and concentration. Solution pH, ionic strength, cofactors, chemical chaperones, and osmolytes perturb the chemical potential and induce further changes in structure, stability, and function. At present, no single analytical technique can monitor these effects in a single measurement. Mass spectrometry and ion mobility-mass spectrometry play increasingly essential roles in studies of proteins, protein complexes, and even membrane protein complexes; however, with few exceptions, the effects of the solution temperature on the stability and structure(s) of analytes have not been thoroughly investigated.Here, we describe a new variable-temperature electrospray ionization (vT-ESI) source that utilizes a thermoelectric chip to cool and heat the solution contained within the static ESI emitter. This design allows for solution temperatures to be varied from ∼5 to 98 °C with short equilibration times (<2 min) between precisely controlled temperature changes. The performance of the apparatus for vT-ESI-mass spectrometry and vT-ESI-ion mobility-mass spectrometry studies of cold-and heat-folding reactions is demonstrated using ubiquitin and frataxin. Instrument performance for studies on temperature-dependent ligand binding is shown using the chaperonin GroEL.

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Section: ■ Results and Discussionmentioning

confidence: 99%

Variable-Temperature Electrospray Ionization for Temperature-Dependent Folding/Refolding Reactions of Proteins and Ligand Binding

et al. 2021

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“…Instrument development for native‐MS is largely driven by needs to understand the structural details of large protein complexes, viz . solvent manipulation (Shi et al, 2014), effects of protein‐protein interactions (Cong et al, 2016), PTMs (Fornelli et al, 2020), stoichiometry (Kaltashov & Mohimen, 2005; Zhou & Wysocki, 2014; Shirzadeh et al, 2019; Stiving et al, 2019; VanAernum et al, 2019), and ligand binding events (Hyung, Robinson, & Ruotolo, 2009; Allison et al, 2015; Cong et al, 2016; Marchand et al, 2018), investigations of which are made possible by recent advances in MS instrumentation (Giles et al, 2019 Poltash et al, 2020). Advances in IM‐MS technology and experiments were accelerated by the readily adopted Waters SYNAPT platform.…”

Section: Next‐generation Im‐ms Technologies For Studies Of Large Protmentioning

confidence: 99%

“…The versatile SYNAPT platform allows for pre‐IM ion mass selection allowing for tandem MS experiments that can be further interrogated by the novel traveling‐wave IM with additional fragmentation, that is, CID (Mitchell Wells & McLuckey, 2005), SID (Zhou & Wysocki, 2014; Stiving et al, 2019; VanAernum et al, 2019), and electron transfer/capture dissociation (Zhurov et al, 2013). However, perturbations in the structure of native proteins are difficult to study, because the changes in structure may lead to small changes in CCS that are unresolvable on commercial instrumentation (Poltash et al, 2020). To understand the relatively minute structural perturbations for large proteins and protein complexes, new instrumentation needs to be developed to overcome the limitations of commercial instrumentation in terms of insufficient resolving power (RP), that is, peak centroid/separation of two closely spaced peaks) in the IM and m / z domains (RP IM and RP m / z , respectively; Poltash et al, 2020).…”

Section: Next‐generation Im‐ms Technologies For Studies Of Large Protmentioning

confidence: 99%

“…However, perturbations in the structure of native proteins are difficult to study, because the changes in structure may lead to small changes in CCS that are unresolvable on commercial instrumentation (Poltash et al, 2020). To understand the relatively minute structural perturbations for large proteins and protein complexes, new instrumentation needs to be developed to overcome the limitations of commercial instrumentation in terms of insufficient resolving power (RP), that is, peak centroid/separation of two closely spaced peaks) in the IM and m / z domains (RP IM and RP m / z , respectively; Poltash et al, 2020). For example, as shown for TTR, a RP m / z of 840 is needed to resolve Zn binding to the intact tetrameric protein that was previously hidden.…”

Section: Next‐generation Im‐ms Technologies For Studies Of Large Protmentioning

confidence: 99%

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The Ims Paradox: A Perspective on Structural Ion Mobility‐mass Spectrometry

McCabe

Hebert

Shirzadeh

et al. 2020

Mass Spectrometry Reviews

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Studies of large proteins, protein complexes, and membrane protein complexes pose new challenges, most notably the need for increased ion mobility (IM) and mass spectrometry (MS) resolution. This review covers evolutionary developments in IM‐MS in the authors' and key collaborators' laboratories with specific focus on developments that enhance the utility of IM‐MS for structural analysis. IM‐MS measurements are performed on gas phase ions, thus “structural IM‐MS” appears paradoxical—do gas phase ions retain their solution phase structure? There is growing evidence to support the notion that solution phase structure(s) can be retained by the gas phase ions. It should not go unnoticed that we use “structures” in this statement because an important feature of IM‐MS is the ability to deal with conformationally heterogeneous systems, thus providing a direct measure of conformational entropy. The extension of this work to large proteins and protein complexes has motivated our development of Fourier‐transform IM‐MS instruments, a strategy first described by Hill and coworkers in 1985 (Anal Chem, 1985, 57, pp. 402–406) that has proved to be a game‐changer in our quest to merge drift tube (DT) and ion mobility and the high mass resolution orbitrap MS instruments. DT‐IMS is the only method that allows first‐principles determinations of rotationally averaged collision cross sections (CSS), which is essential for studies of biomolecules where the conformational diversities of the molecule precludes the use of CCS calibration approaches. The Fourier transform‐IM‐orbitrap instrument described here also incorporates the full suite of native MS/IM‐MS capabilities that are currently employed in the most advanced native MS/IM‐MS instruments. © 2020 John Wiley & Sons Ltd. Mass Spec Rev

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“…A high‐resolution mass spectrum revealing the microheterogeneity of Asialo‐HP glycoprotein is shown in Figure 10B as an example. Very recently, high‐resolution native MS and IM were successfully coupled allowing separation of the gas phase structures of highly resolved protein complex' populations 192 . In future, many more applications of high‐resolution native MS are anticipated.…”

Section: Recent Advances Enable High‐resolution Native Msmentioning

confidence: 99%

Native mass spectrometry—A valuable tool in structural biology

Barth

Schmidt

2020

J Mass Spectrom

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Proteins and the complexes they form with their ligands are the players of cellular action. Their function is directly linked with their structure making the structural analysis of protein-ligand complexes essential. Classical techniques of structural biology include X-ray crystallography, nuclear magnetic resonance spectroscopy and recently distinguished cryo-electron microscopy. However, protein-ligand complexes are often dynamic and heterogeneous and consequently challenging for these techniques. Alternative approaches are therefore needed and gained importance during the last decades. One alternative is native mass spectrometry, which is the analysis of intact protein complexes in the gas phase. To achieve this, sample preparation and instrument conditions have to be optimised. Native mass spectrometry then reveals stoichiometry, protein interactions and topology of protein assemblies. Advanced techniques such as ion mobility and high-resolution mass spectrometry further add to the range of applications and deliver information on shape and microheterogeneity of the complexes. In this tutorial, we explain the basics of native mass spectrometry including sample requirements, instrument modifications and interpretation of native mass spectra. We further discuss the developments of native mass spectrometry and provide example spectra and applications.

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Native IM-Orbitrap MS: Resolving what was hidden

Cited by 39 publications

References 58 publications

Variable-Temperature Electrospray Ionization for Temperature-Dependent Folding/Refolding Reactions of Proteins and Ligand Binding

Variable-Temperature Electrospray Ionization for Temperature-Dependent Folding/Refolding Reactions of Proteins and Ligand Binding

The Ims Paradox: A Perspective on Structural Ion Mobility‐mass Spectrometry

Native mass spectrometry—A valuable tool in structural biology

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