Exploring an Orbitrap Analyzer for the Characterization of Intact Antibodies by Native Mass Spectrometry

2012 ◽  
Vol 51 (52) ◽  
pp. 12992-12996 ◽  
Author(s):  
Sara Rosati ◽  
Rebecca J. Rose ◽  
Natalie J. Thompson ◽  
Esther van Duijn ◽  
Eugen Damoc ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Native Mass Spectrometry ◽  
Orbitrap Analyzer

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

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).

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

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.

scholarly journals Characterization of trimethoprim resistant E. coli dihydrofolate reductase mutants by mass spectrometry and inhibition by propargyl-linked antifolates

2017 ◽  
Vol 8 (5) ◽  
pp. 4062-4072 ◽  
Author(s):  
Michael Cammarata ◽  
Ross Thyer ◽  
Michael Lombardo ◽  
Amy Anderson ◽  
Dennis Wright ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Size Exclusion Chromatography ◽  
Dihydrofolate Reductase ◽  
Native Mass Spectrometry ◽  
Size Exclusion ◽  
E Coli ◽  
Exclusion Chromatography

Native mass spectrometry, size exclusion chromatography, and kinetic assays were employed to study trimethoprim resistance in E. coli caused by mutations P21L and W30R of dihydrofolate reductase.

scholarly journals Detailed Characterization of Monoclonal Antibody Receptor Interaction Using Affinity Liquid Chromatography Hyphenated to Native Mass Spectrometry

2017 ◽  
Vol 89 (10) ◽  
pp. 5404-5412 ◽  
Author(s):  
Rabah Gahoual ◽  
Anna-Katharina Heidenreich ◽  
Govert W. Somsen ◽  
Patrick Bulau ◽  
Dietmar Reusch ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Monoclonal Antibody ◽  
Liquid Chromatography ◽  
Native Mass Spectrometry ◽  
Receptor Interaction ◽  
Detailed Characterization

scholarly journals Characterization of the T4 gp32–ssDNA complex by native, cross-linking, and ultraviolet photodissociation mass spectrometry

2021 ◽  
Author(s):  
Molly S. Blevins ◽  
Jada N. Walker ◽  
Jeffrey M. Schaub ◽  
Ilya J. Finkelstein ◽  
Jennifer S. Brodbelt
Keyword(s):  
Mass Spectrometry ◽  
Native Mass Spectrometry ◽  
Cross Linking ◽  
Binding Interactions ◽  
Ultraviolet Photodissociation

Ultraviolet photodissociation and native mass spectrometry allow characterization of the formation and binding interactions of protein-ssDNA complexes.

scholarly journals Rapid characterization of secreted recombinant proteins by native mass spectrometry

2018 ◽  
Vol 1 (1) ◽  
Author(s):  
Gili Ben-Nissan ◽  
Shay Vimer ◽  
Shira Warszawski ◽  
Aliza Katz ◽  
Meital Yona ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Recombinant Proteins ◽  
Native Mass Spectrometry ◽  
Rapid Characterization

scholarly journals Native mass spectrometry and ion mobility characterization of trastuzumab emtansine, a lysine-linked antibody drug conjugate

2015 ◽  
Vol 24 (8) ◽  
pp. 1210-1223 ◽  
Author(s):  
Julien Marcoux ◽  
Thierry Champion ◽  
Olivier Colas ◽  
Elsa Wagner-Rousset ◽  
Nathalie Corvaïa ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Ion Mobility ◽  
Native Mass Spectrometry ◽  
Trastuzumab Emtansine ◽  
Antibody Drug Conjugate ◽  
Drug Conjugate ◽  
Antibody Drug
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