Native Mass Spectrometry, Ion Mobility, Electron-Capture Dissociation, and Modeling Provide Structural Information for Gas-Phase Apolipoprotein E Oligomers

Antibody-drug conjugates (ADCs) are biotherapeutics consisting of a tumor-targeting monoclonal antibody (mAb) linked covalently to a cytotoxic drug. Early generation ADCs were predominantly obtained through non-selective conjugation methods based on lysine and cysteine residues, resulting in heterogeneous populations with varying drug-to-antibody ratios (DAR). Site-specific conjugation is one of the current challenges in ADC development, allowing for controlled conjugation and production of homogeneous ADCs. We report here the characterization of a site-specific DAR2 ADC generated with the GlyCLICK three-step process, which involves glycan-based enzymatic remodeling and click chemistry, using state-of-the-art native mass spectrometry (nMS) methods. The conjugation process was monitored with size exclusion chromatography coupled to nMS (SEC-nMS), which offered a straightforward identification and quantification of all reaction products, providing a direct snapshot of the ADC homogeneity. Benefits of SEC-nMS were further demonstrated for forced degradation studies, for which fragments generated upon thermal stress were clearly identified, with no deconjugation of the drug linker observed for the T-GlyGLICK-DM1 ADC. Lastly, innovative ion mobility-based collision-induced unfolding (CIU) approaches were used to assess the gas-phase behavior of compounds along the conjugation process, highlighting an increased resistance of the mAb against gas-phase unfolding upon drug conjugation. Altogether, these state-of-the-art nMS methods represent innovative approaches to investigate drug loading and distribution of last generation ADCs, their evolution during the bioconjugation process and their impact on gas-phase stabilities. We envision nMS and CIU methods to improve the conformational characterization of next generation-empowered mAb-derived products such as engineered nanobodies, bispecific ADCs or immunocytokines.

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Electron capture dissociation and drift tube ion mobility-mass spectrometry coupled with site directed mutations provide insights into the conformational diversity of a metamorphic protein

Physical Chemistry Chemical Physics ◽

10.1039/c4cp05136j ◽

2015 ◽

Vol 17 (16) ◽

pp. 10538-10550 ◽

Cited By ~ 10

Author(s):

Sophie R. Harvey ◽

Massimiliano Porrini ◽

Robert C. Tyler ◽

Cait E. MacPhee ◽

Brian F. Volkman ◽

...

Keyword(s):

Mass Spectrometry ◽

Electron Capture ◽

Electron Capture Dissociation ◽

Ion Mobility ◽

Drift Tube ◽

Ion Mobility Mass Spectrometry ◽

Top Down ◽

Conformational Diversity ◽

Metamorphic Protein

Ion mobility mass spectrometry can be combined with data from top-down sequencing to discern adopted conformations of proteins in the absence of solvent.

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Exploring the conformational landscape of a lanthipeptide synthetase using native mass spectrometry

10.1101/2020.09.01.277863 ◽

2020 ◽

Author(s):

Nuwani W. Weerasinghe ◽

Yeganeh Habibi ◽

Kevin A. Uggowitzer ◽

Christopher J. Thibodeaux

Keyword(s):

Mass Spectrometry ◽

Gas Phase ◽

Conformational Changes ◽

Ion Mobility ◽

Peptide Binding ◽

Native Mass Spectrometry ◽

Order Structure ◽

Conformational Sampling ◽

Precursor Peptide ◽

Conformational Landscape

AbstractLanthipeptides are ribosomally-synthesized and post-translationally modified peptide (RiPP) natural products that are biosynthesized in a multistep maturation process by enzymes (lanthipeptide synthetases) that possess relaxed substrate specificity. Recent evidence has suggested that some lanthipeptide synthetases are structurally dynamic enzymes that are allosterically activated by precursor peptide binding, and that conformational sampling of the enzyme-peptide complex may play an important role in defining the efficiency and sequence of biosynthetic events. These “biophysical” processes, while critical for defining the activity and function of the synthetase, remain very challenging to study with existing methodologies. Herein, we show that native nanoelectrospray ionization coupled to ion mobility mass spectrometry (nanoESI-IM-MS) provides a powerful and sensitive means for investigating the conformational landscapes and intermolecular interactions of lanthipeptide synthetases. Namely, we demonstrate that the class II lanthipeptide synthetase (HalM2) and its non-covalent complex with the cognate HalA2 precursor peptide can be delivered into the gas phase in a manner that preserves native structures and intermolecular enzyme-peptide contacts. Moreover, gas phase ion mobility studies of the natively-folded ions demonstrate that peptide binding and mutations to dynamic structural elements of HalM2 alter the conformational landscape of the enzyme, and that the precursor peptide itself exhibits higher order structure in the mass spectrometer. Cumulatively, these data support previous claims that lanthipeptide synthetases are structurally dynamic enzymes that undergo functionally relevant conformational changes in response to precursor peptide binding. This work establishes nanoESI-IM-MS as a versatile approach for unraveling the relationships between protein structure and biochemical function in RiPP biosynthetic systems.

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Electron-capture dissociation and ion mobility mass spectrometry for characterization of the hemoglobin protein assembly

Protein Science ◽

10.1002/pro.2712 ◽

2015 ◽

Vol 24 (8) ◽

pp. 1325-1332 ◽

Cited By ~ 20

Author(s):

Weidong Cui ◽

Hao Zhang ◽

Robert E. Blankenship ◽

Michael L. Gross

Keyword(s):

Mass Spectrometry ◽

Electron Capture ◽

Electron Capture Dissociation ◽

Ion Mobility ◽

Protein Assembly ◽

Ion Mobility Mass Spectrometry

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Structural Analysis of 14-3-3-ζ-Derived Phosphopeptides Using Electron Capture Dissociation Mass Spectrometry, Traveling Wave Ion Mobility Spectrometry, and Molecular Modeling

The Journal of Physical Chemistry B ◽

10.1021/acs.jpcb.9b08506 ◽

2019 ◽

Vol 124 (3) ◽

pp. 461-469 ◽

Cited By ~ 1

Author(s):

Anna L. Simmonds ◽

Andrea F. Lopez-Clavijo ◽

Peter J. Winn ◽

David H. Russell ◽

Iain B. Styles ◽

...

Keyword(s):

Mass Spectrometry ◽

Molecular Modeling ◽

Structural Analysis ◽

Electron Capture ◽

Electron Capture Dissociation ◽

Ion Mobility Spectrometry ◽

Ion Mobility ◽

Traveling Wave ◽

Traveling Wave Ion Mobility

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Conformational footprinting of proteins using a combination of top-down electron transfer dissociation and ion mobility

10.1101/283796 ◽

2018 ◽

Author(s):

Albert Konijnenberg ◽

Jinyu Li ◽

Johny Habchi ◽

Marion Dosnon ◽

Giulia Rossetti ◽

...

Keyword(s):

Protein Structure ◽

Gas Phase ◽

Ion Mobility ◽

Measles Virus ◽

Electron Transfer Dissociation ◽

Structural Information ◽

Native Mass Spectrometry ◽

Top Down ◽

Wide Range ◽

Protein Ensembles

AbstractIn recent years native mass spectrometry has been increasingly employed to study protein structure. As such a thorough understanding of the effect of the gas-phase on protein structure is becoming increasingly important. We show how a combination of top-down ETD and ion mobility can be used to probe the gas-phase structure of heterogeneous protein ensembles. By applying collisional activation to the non-covalently bound ETD products after IM separation, the peptide fragments can be released while maintaining the conformational information of the protein ion. We studied the unknown gas-phase structures of the measles virus (MeV) phosphoprotein X domain (PXD), which shows a wide range of different conformations in the gas-phase. We then generated structural models by state-of-the-art gas-phase steered molecular dynamics, which we verified using restraints from ion mobility and the fragment patterns observed. Our findings illustrate the applicability of ETD for obtaining conformational specific structural information on heterogeneous protein ensembles.

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DNA and RNA Telomeric G-Quadruplexes: What Topology Features Can be Inferred from Ion Mobility Mass Spectrometry?

10.26434/chemrxiv.8246975 ◽

2019 ◽

Author(s):

Valentina D’Atri ◽

Valerie Gabelica

Keyword(s):

Mass Spectrometry ◽

Gas Phase ◽

Ion Mobility Spectrometry ◽

Ion Mobility ◽

Native Mass Spectrometry ◽

Cation Binding ◽

Telomeric Dna ◽

Dna And Rna ◽

G Quadruplex ◽

Multiple Structures

Maintenance of the telomeres is key to chromosome integrity and cell proliferation. The G-quadruplex structures formed by telomeric DNA and RNA (TTAGGG and UUAGGG repeats, respectively) are key to this process. However, because these sequences are particularly polymorphic, solving high-resolution structures is not always possible, and there is a need for new methodologies to characterize the multiple structures coexisting in solution. In this context, we evaluated whether ion mobility spectrometry coupled to native mass spectrometry could help separate and assign the G-quadruplex topologies. We explored the circular dichroism spectra, multimer formation, cation binding, and ion mobility spectra of several 4-repeat and 8-repeat telomeric DNA and RNA sequences, both in NH4+ and in K+. In 1 mM K+ and 100 mM trimethylammonium acetate, all RNAs fold intramolecularly (no multimer). In 8-repeat sequences, the subunits are not independent: in DNA the first subunit disfavors the folding of the second one, whereas in RNA the two subunits fold cooperatively via cation-mediated stacking. Ion mobility spectrometry shows that gas-phase structures keep a memory of the solution ones, but not identical. At the native charge states, the loops can rearrange in a variety of ways (unless they are constrained by pre-formed hydrogen bonds), thereby wrapping the core and masking the strand arrangements. Our study highlights that, to progress towards structural assignment from IM-MS experiments, deeper understanding of the solution-to-gas-phase rearrangement mechanisms is warranted.

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