How can native mass spectrometry contribute to characterization of biomacromolecular higher-order structure and interactions?

Background: Biotherapeutics, particularly monoclonal antibodies (mAbs), are a maturing class of drugs capable of treating a wide range of diseases. Therapeutic function and solutionstability are linked to the proper three-dimensional organization of the primary sequence into Higher Order Structure (HOS) as well as the timescales of protein motions (dynamics). Methods that directly monitor protein HOS and dynamics are important for mapping therapeutically relevant protein-protein interactions and assessing properly folded structures. Irreversible covalent protein footprinting Mass Spectrometry (MS) tools, such as site-specific amino acid labeling and hydroxyl radical footprinting are analytical techniques capable of monitoring the side chain solvent accessibility influenced by tertiary and quaternary structure. Here we discuss the methodology, examples of biotherapeutic applications, and the future directions of irreversible covalent protein footprinting MS in biotherapeutic research and development. Conclusion: Bottom-up mass spectrometry using irreversible labeling techniques provide valuable information for characterizing solution-phase protein structure. Examples range from epitope mapping and protein-ligand interactions, to probing challenging structures of membrane proteins. By paring these techniques with hydrogen-deuterium exchange, spectroscopic analysis, or static-phase structural data such as crystallography or electron microscopy, a comprehensive understanding of protein structure can be obtained.

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DNA G-quadruplexes for native mass spectrometry in potassium: a database of validated structures in electrospray-compatible conditions

Nucleic Acids Research ◽

10.1093/nar/gkab039 ◽

2021 ◽

Author(s):

Anirban Ghosh ◽

Eric Largy ◽

Valérie Gabelica

Keyword(s):

Mass Spectrometry ◽

Drug Targets ◽

Native Mass Spectrometry ◽

Drug Binding ◽

Quadruplex Dna ◽

Dna Structures ◽

Nmr Structures ◽

G Quadruplex ◽

Screening Assays

Abstract G-quadruplex DNA structures have become attractive drug targets, and native mass spectrometry can provide detailed characterization of drug binding stoichiometry and affinity, potentially at high throughput. However, the G-quadruplex DNA polymorphism poses problems for interpreting ligand screening assays. In order to establish standardized MS-based screening assays, we studied 28 sequences with documented NMR structures in (usually ∼100 mM) potassium, and report here their circular dichroism (CD), melting temperature (Tm), NMR spectra and electrospray mass spectra in 1 mM KCl/100 mM trimethylammonium acetate. Based on these results, we make a short-list of sequences that adopt the same structure in the MS assay as reported by NMR, and provide recommendations on using them for MS-based assays. We also built an R-based open-source application to build and consult a database, wherein further sequences can be incorporated in the future. The application handles automatically most of the data processing, and allows generating custom figures and reports. The database is included in the g4dbr package (https://github.com/EricLarG4/g4dbr) and can be explored online (https://ericlarg4.github.io/G4_database.html).

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Mass Spectrometry-Based Fast Photochemical Oxidation of Proteins (FPOP) for Higher Order Structure Characterization

Accounts of Chemical Research ◽

10.1021/acs.accounts.7b00593 ◽

2018 ◽

Vol 51 (3) ◽

pp. 736-744 ◽

Cited By ~ 39

Author(s):

Ke Sherry Li ◽

Liuqing Shi ◽

Michael L. Gross

Keyword(s):

Mass Spectrometry ◽

Higher Order ◽

Structure Characterization ◽

Photochemical Oxidation ◽

Order Structure

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State-of-the-Art Native Mass Spectrometry and Ion Mobility Methods to Monitor Homogeneous Site-Specific Antibody-Drug Conjugates Synthesis

Pharmaceuticals ◽

10.3390/ph14060498 ◽

2021 ◽

Vol 14 (6) ◽

pp. 498

Author(s):

Evolène Deslignière ◽

Anthony Ehkirch ◽

Bastiaan L. Duivelshof ◽

Hanna Toftevall ◽

Jonathan Sjögren ◽

...

Keyword(s):

Mass Spectrometry ◽

Gas Phase ◽

Ion Mobility ◽

State Of The Art ◽

Native Mass Spectrometry ◽

Site Specific ◽

Drug Conjugates ◽

Conjugation Process ◽

Antibody Drug

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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