Application of the Exactive Plus EMR for automated protein–ligand screening by non‐covalent mass spectrometry

Maple, Hannah J.; Scheibner, Olaf; Baumert, Mark; Allen, Mark; Taylor, Richard J. K.; Garlish, Rachel A.; Bromirski, Maciej; Burnley, Rebecca J.

doi:10.1002/rcm.6925

Cited by 26 publications

(32 citation statements)

References 43 publications

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“…To better meet the requirements necessary for the analysis of intact antibodies, antibodydrug conjugates, large protein complexes, and native proteins, an extended mass range Orbitrap instrument was developed with improved sensitivity to these analytes (47)(48)(49)(50). These mass spectrometers have been optimized for improved transmission of higher mass-to-charge-ratio ions, enabling detection up to m/z 20,000.…”

Section: Improving Intact Protein and Top-down Analysismentioning

confidence: 99%

Evolution of Orbitrap Mass Spectrometry Instrumentation

Eliuk

Makarov²

2015

Annual Rev. Anal. Chem.

377

329

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We discuss the evolution of Orbitrap mass spectrometry (MS) from its birth in the late 1990s to its current role as one of the most prominent techniques for MS. The Orbitrap mass analyzer is the first high-performance mass analyzer that employs trapping of ions in electrostatic fields. Tight integration with the ion injection process enables the high-resolution, mass accuracy, and sensitivity that have become essential for addressing analytical needs in numerous areas of research, as well as in routine analysis. We examine three major families of instruments (related to the LTQ Orbitrap, Q Exactive, and Orbitrap Fusion mass spectrometers) in the context of their historical development over the past ten eventful years. We discuss as well future trends and perspectives of Orbitrap MS. We illustrate the compelling potential of Orbitrap-based mass spectrometers as (ultra) high-resolution platforms, not only for high-end proteomic applications, but also for routine targeted analysis.

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Section: Improving Intact Protein and Top-down Analysismentioning

confidence: 99%

Evolution of Orbitrap Mass Spectrometry Instrumentation

Eliuk

Makarov²

2015

Annual Rev. Anal. Chem.

377

329

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“…Native mass spectrometry (MS) is an emerging biophysical technique for studying protein structure and function. , In native MS, protein complexes in a volatile, nondenaturing solution (typically ammonium acetate) are ionized using nanoelectrospray ionization , and the instrument is tuned to preserve protein structure and noncovalent interactions in the mass spectrometer. − This unique ability has enabled the analysis of direct ligand binding, protein dynamics and topology, and subunit stoichiometry and measurement of thermodynamics and binding affinities. − More recently, high-resolution native mass spectrometry has been used to characterize the binding of small molecules such as drugs, nucleotides, lipids, metals, and small enzyme cofactors. ,, Ion mobility (IM) is a gas phase electrophoretic separation of molecules based on the size and shape of ions, and when it is combined with MS, it can provide structural information by measuring the rotationally averaged collision cross section (CCS). Native IM-MS can monitor direct molecular interactions of molecules with protein complexes and also provide unparalleled insight into the purity of protein samples. , …”

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confidence: 99%

Intrinsic GTPase Activity of K-RAS Monitored by Native Mass Spectrometry

et al. 2019

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Mutations in RAS are associated with many different cancers and have been a therapeutic target for more than three decades. RAS cycles from an active to inactive state by both intrinsic and GTPase-activating protein (GAP)-stimulated hydrolysis. The activated enzyme interacts with downstream effectors, leading to tumor proliferation. Mutations in RAS associated with cancer are insensitive to GAP, and the rate of inactivation is limited to their intrinsic hydrolysis rate. Here, we use high-resolution native mass spectrometry (MS) to determine the kinetics and transition state thermodynamics of intrinsic hydrolysis for K-RAS and its oncogenic mutants. MS data reveal heterogeneity where both 2′-deoxy and 2′-hydroxy forms of GDP (guanosine diphosphate) and GTP (guanosine triphosphate) are bound to the recombinant enzyme. Intrinsic GTPase activity is directly monitored by the loss in mass of K-RAS bound to GTP, which corresponds to the release of phosphate. The rates determined from MS are in direct agreement with those measured using an established solution-based assay. Our results show that the transition state thermodynamics for the intrinsic GTPase activity of K-RAS is both enthalpically and entropically unfavorable. The oncogenic mutants G12C, Q61H, and G13D unexpectedly exhibit a 2′-deoxy GTP intrinsic hydrolysis rate higher than that for GTP.

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“…Alternative nanoESI microfluidic devices, e.g. the Triversa Nanomate from Advion, have been developed, combining capabilities of sample preparation such as ligand addition to the protein and temperature-controlled incubation to reproducible nanoESI injection and nMS analysis 43 . Benefits of such systems is that using individual nozzles for each ligand/mixture of ligand prevents from cross contamination of capillaries or columns.…”

Section: Sec-nms Is Suitable For a Variety Of Noncovalent Complexes Analysesmentioning

confidence: 99%

Pushing the limits of native MS: Online SEC-native MS for structural biology applications

Deslignière

Ley

Bourguet

et al. 2021

International Journal of Mass Spectrometry

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Native mass spectrometry (nMS) is now widely applied to investigate non-covalently assembled biomolecule complexes. nMS requires the use of near-neutral pH and volatile buffers to preserve the native state of proteins. However, buffer exchange into nMS-compatible solvent is usually performed manually, which results in a time-consuming and tedious process, thus appearing as a major drawback for nMS analysis. Conversely, online coupling of size exclusion chromatography (SEC) to nMS affords a fast-automated and improved desalting, but also provides an additional dimension of separation for complex protein mixtures. We illustrate here the benefits of SEC-nMS compared to manual offline desalting for the characterization of a wide variety of biological systems, ranging from multiprotein assemblies, protein-ligands and protein-nucleic acid complexes, to proteins in a detergent environment. We then highlight the potential of the coupling to further integrate ion mobility while preserving the native conformations of proteins, allowing for rapid collision cross section measurement and even collision-induced unfolding experiments. Finally, we show that online SEC coupling can also serve as the basis for multidimensional non-denaturing liquid chromatography (LC) workflows, with the SEC acting as a fast desalting device, helping to achieve first dimension LC separation in optimal chromatographic conditions while being compatible with further nMS analysis.

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Application of the Exactive Plus EMR for automated protein–ligand screening by non‐covalent mass spectrometry

Cited by 26 publications

References 43 publications

Evolution of Orbitrap Mass Spectrometry Instrumentation

Evolution of Orbitrap Mass Spectrometry Instrumentation

Intrinsic GTPase Activity of K-RAS Monitored by Native Mass Spectrometry

Pushing the limits of native MS: Online SEC-native MS for structural biology applications

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