Fourier Transform-Ion Mobility-Orbitrap Mass Spectrometer: A Next-Generation Instrument for Native Mass Spectrometry

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

doi:10.1021/acs.analchem.8b02463

Cited by 72 publications

(103 citation statements)

References 67 publications

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“…We next performed measurements on a custom-built Orbitrap Ultra High Mass Range (UHMR) modified with REIS and a 1.5 meter periodic focusing drift tube operating in the FT-mode. 43 The first principles CCS for the K 2P 4.1a was is in agreement with those calculated for the crystal structure of K 2P 4.1 ( Extended Data Fig. 1E – F ).…”

Section: Resultssupporting

confidence: 83%

Selective regulation of human TRAAK channels by biologically active phospholipids

et al. 2020

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TRAAK is an ion channel from the two-pore domain potassium (K 2P ) channel family with roles in maintaining the resting membrane potential and fast action potential conduction. Regulated by a wide range of physical and chemical stimuli, the affinity and selectivity of K 2P 4.1 towards lipids remains poorly understood. Here we show the two isoforms of K 2P 4.1 have distinct binding preferences for lipids dependent on acyl chain length and position on the glycerol backbone. Unexpectedly, the channel can also discriminate the fatty acid linkage at the sn -1 position. Of the 33 lipids interrogated using native mass spectrometry, phosphatidic acid (PA) had the lowest equilibrium dissociation constants for both isoforms of K 2P 4.1. Liposome potassium flux assays with K 2P 4.1 reconstituted in defined lipid environments show that those containing PA activate the channel in a dose-dependent fashion. Our results begin to define the molecular requirements for the specific binding of lipids to K 2P 4.1.

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Section: Resultssupporting

confidence: 83%

Selective regulation of human TRAAK channels by biologically active phospholipids

et al. 2020

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“…We also acquired data on a higher mass resolution Orbitrap mass spectrometer 48 equipped with a custom reverse-entry ion source (REIS-Orbitrap) 49 , 50 that yielded sharp mass spectral peaks with addition of mass as small as 80 Da resolved (Fig. 1d ).…”

Section: Resultsmentioning

confidence: 99%

Selective binding of a toxin and phosphatidylinositides to a mammalian potassium channel

Liu

LoCaste

et al. 2019

Nat Commun

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G-protein-gated inward rectifying potassium channels (GIRKs) require Gβγ subunits and phosphorylated phosphatidylinositides (PIPs) for gating. Although studies have provided insight into these interactions, the mechanism of how these events are modulated by Gβγ and the binding affinity between PIPs and GIRKs remains poorly understood. Here, native ion mobility mass spectrometry is employed to directly monitor small molecule binding events to mouse GIRK2. GIRK2 binds the toxin tertiapin Q and PIPs selectively and with significantly higher affinity than other phospholipids. A mutation in GIRK2 that causes a rotation in the cytoplasmic domain, similarly to Gβγ-binding to the wild-type channel, revealed differences in the selectivity towards PIPs. More specifically, PIP isoforms known to weakly activate GIRKs have decreased binding affinity. Taken together, our results reveal selective small molecule binding and uncover a mechanism by which rotation of the cytoplasmic domain can modulate GIRK•PIP interactions.

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“…Current commercial IM‐MS instruments suffer from low MS resolution ( R m / z ) in the TOF with only recent implementations of high‐resolution TOFs (e.g., Waters SELECT Series Cyclic IMS or Agilent 6545XT Q‐TOF MS, but the latter lacks IM capabilities) (Giles et al, 2019). Alternatively, IM can be coupled to a high‐resolution orbitrap platform, but this introduces a duty‐cycle mismatch since the orbitrap operates on a timescale of hundreds of milliseconds (Clowers & Hill, 2005; Szumlas, Ray, & Hieftje, 2006; Morrison, Siems, & Clowers, 2016; Poltash et al, 2018). This problem may be mitigated by supplementing the gate at the entrance of the DT with a second gate implemented post‐IM to isolate a single drift time of interest to be transferred for mass analysis; this process is then iterated over the entire drift time window.…”

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

confidence: 99%

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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Fourier Transform-Ion Mobility-Orbitrap Mass Spectrometer: A Next-Generation Instrument for Native Mass Spectrometry

Cited by 72 publications

References 67 publications

Selective regulation of human TRAAK channels by biologically active phospholipids

Selective regulation of human TRAAK channels by biologically active phospholipids

Selective binding of a toxin and phosphatidylinositides to a mammalian potassium channel

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

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