Mass spectrometry of intact membrane protein complexes

Laganowsky, Arthur; Reading, Eamonn; Hopper, Jonathan T. S.; Robinson, Carol V.

doi:10.1038/nprot.2013.024

Cited by 354 publications

(453 citation statements)

References 24 publications

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“…IM-MS has proven exceptionally useful for providing insights into the structure of membrane proteins, such as subunit stoichiometries and phospholipid binding (26,27). Current methodology depends strongly on the apparently protective capabilities of detergent micelles to transfer membrane proteins intact into the vacuum of the mass spectrometer (26,28), followed by the removal of these detergents by collisional activation. However, the energy required to rid the membrane protein of its detergent cover often results in the loss of structural integrity (28,29).…”

Section: Resultsmentioning

confidence: 99%

“…Current methodology depends strongly on the apparently protective capabilities of detergent micelles to transfer membrane proteins intact into the vacuum of the mass spectrometer (26,28), followed by the removal of these detergents by collisional activation. However, the energy required to rid the membrane protein of its detergent cover often results in the loss of structural integrity (28,29). To enable analysis of native MscL structure using IM-MS (22-25), we addressed this problem and screened several detergents for their ability to preserve and…”

Section: Resultsmentioning

confidence: 99%

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Global structural changes of an ion channel during its gating are followed by ion mobility mass spectrometry

Konijnenberg

Yilmaz²,

Ingólfsson

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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Mechanosensitive ion channels are sensors probing membrane tension in all species; despite their importance and vital role in many cell functions, their gating mechanism remains to be elucidated. Here, we determined the conditions for releasing intact mechanosensitive channel of large conductance (MscL) proteins from their detergents in the gas phase using native ion mobilitymass spectrometry (IM-MS). By using IM-MS, we could detect the native mass of MscL from Escherichia coli, determine various global structural changes during its gating by measuring the rotationally averaged collision cross-sections, and show that it can function in the absence of a lipid bilayer. We could detect global conformational changes during MscL gating as small as 3%. Our findings will allow studying native structure of many other membrane proteins.ion mobility mass spectrometry | MscL | membrane proteins | structure function | ion channel gating O ne of the best candidates to explore the gating of mechanosensitive channels is the mechanosensitive channel of large conductance (MscL) from Escherichia coli. The crystal structure of MscL in its closed/nearly closed state from Mycobacterium tuberculosis revealed this channel as a homopentamer (1). Each subunit has a cytoplasmic N-and C-terminal domain as well as two α-helical transmembrane (TM) domains, TM1 and TM2, which are connected by a periplasmic loop. The five TM1 helices form the pore and the more peripheral TM2 helices interact with the lipid bilayer.MscL detects changes in membrane tension invoked by a hypoosmotic shock and couples the tension sensing directly to large conformational changes (1, 2). On the basis of a large body of structural and theoretical data, numerous gating models of MscL have been proposed (3-9). These models agree upon (i) the hydrophobic pore constriction of the channel and (ii) the channel opens by an iris-like rotation-i.e., a tilting and outward movement of transmembrane helices that make the channel wider and shorter (5). This mechanism is supported by patchclamp (10), disulfide cross-linking (11), FRET spectroscopy (12), and site-directed spin labeling EPR experiments (6, 7), as well as computational studies (13-15). So far, direct experimental results have only been observed for short-range local structural changes, and no measure of the overall global structural changes during channel gating have been reported. Because there is no crystal structure available for the open MscL channel, elucidating overall global structural changes from the onset of channel activation is of utmost importance for our understanding of the gating mechanism of mechanosensitive channels. Here, we provide direct experimental evidence for the key areal changes occurring during channel gating by combining our ability to activate MscL in a controlled manner to different subopen states (16) with a native ion mobility-mass spectrometry (IM-MS) approach. Results and DiscussionDetergents that are Unstable in the Gas Phase Allow Detection of MscL in Its Native Form. Native mass ...

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Global structural changes of an ion channel during its gating are followed by ion mobility mass spectrometry

Konijnenberg

Yilmaz²,

Ingólfsson

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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“…Membrane proteins are notoriously difficult to handle because of their poor solubility, which can be overcome by the use of detergent micelles. However, most ionic detergents are incompatible with ESI, but several nonionic detergents have recently been described to facilitate the analysis of membrane protein complexes with native MS (64). Most notably, recent studies on a variety of ATPases demonstrate how stoichiometry, subunit arrangement, and even lipid and nucleotide binding can be uncovered with native MS (see Figure 3) (63,80).…”

Section: F-and V-type Atpasesmentioning

confidence: 99%

“…Mass analysis with a precision that is routinely below 0.01% has been conducted on various protein complexes. Recent reports include membrane protein complexes (solubilized in detergent micelles) (62)(63)(64), which are particularly challenging for many techniques in structural biology, as well as 18-MDa virus capsids (51), which are thought to be the biggest molecules accessible to the technique with the current state of instrumentation. The resolving power of the technique is often sufficient to monitor posttranslational modifications (such as glycosylation and phosphorylation) and small ligand binding on intact protein complexes up to the megadalton range (18,58,61).…”

Section: Native Mass Spectrometrymentioning

confidence: 99%

Analytical Approaches for Size and Mass Analysis of Large Protein Assemblies

Snijder¹,

Heck

2014

Annual Rev. Anal. Chem.

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Analysis of the size and mass of nanoparticles, whether they are natural biomacromolecular or synthetic supramolecular assemblies, is an important step in the characterization of such molecular species. In recent years, electrospray ionization (ESI) has emerged as a technology through which particles with masses up to 100 MDa can be ionized and transferred into the gas phase, preparing them for accurate mass analysis. Here we review currently used methodologies, with a clear focus on native mass spectrometry (MS). Additional complementary methodologies are also covered, including ion-mobility analysis, nanomechanical mass sensors, and charge-detection MS. The literature discussed clearly demonstrates the great potential of ESI-based methodologies for the size and mass analysis of nanoparticles, including very large naturally occurring protein assemblies. The analytical approaches discussed are powerful tools in not only structural biology, but also nanotechnology. 43

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“…In some cases, structural collapse may take place as well [9,19,38]. These considerations notwithstanding, there are also protein systems that can only be observed when using a substantial level of ion activation (e.g., for shedding detergents prior to IMS and mass analysis [39]). …”

Section: Introductionmentioning

confidence: 99%

Protein Structural Studies by Traveling Wave Ion Mobility Spectrometry: A Critical Look at Electrospray Sources and Calibration Issues

Sun

Vahidi

Sowole

et al. 2015

J. Am. Soc. Mass Spectrom.

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Abstract. The question whether electrosprayed protein ions retain solution-like conformations continues to be a matter of debate. One way to address this issue involves comparisons of collision cross sections (Ω) measured by ion mobility spectrometry (IMS) with Ω values calculated for candidate structures. Many investigations in this area employ traveling wave IMS (TWIMS). It is often implied that nanoESI is more conducive for the retention of solution structure than regular ESI. Focusing on ubiquitin, cytochrome c, myoglobin, and hemoglobin, we demonstrate that Ω values and collisional unfolding profiles are virtually indistinguishable under both conditions. These findings suggest that gas-phase structures and ion internal energies are independent of the type of electrospray source. We also note that TWIMS calibration can be challenging because differences in the extent of collisional activation relative to drift tube reference data may lead to ambiguous peak assignments. It is demonstrated that this problem can be circumvented by employing collisionally heated calibrant ions. Overall, our data are consistent with the view that exposure of native proteins to electrospray conditions can generate kinetically trapped ions that retain solution-like structures on the millisecond time scale of TWIMS experiments.

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Mass spectrometry of intact membrane protein complexes

Cited by 354 publications

References 24 publications

Global structural changes of an ion channel during its gating are followed by ion mobility mass spectrometry

Global structural changes of an ion channel during its gating are followed by ion mobility mass spectrometry

Analytical Approaches for Size and Mass Analysis of Large Protein Assemblies

Protein Structural Studies by Traveling Wave Ion Mobility Spectrometry: A Critical Look at Electrospray Sources and Calibration Issues

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