Synthesis of an Exhaustive Library of Naturally Occurring Galf-Manp and Galp-Manp Disaccharides. Towards Fingerprinting According to the Ring Size by Advanced Mass Spectrometry-Based IM-MS and IRMPD.

Nature offers a huge diversity of glycosidic derivatives. Amongst numerous structural modulations, the nature of the ring size of hexosides may induce significant differences on both biological and physicochemical properties of the glycoconjugate of interest. On this assumption, we expect that small disaccharides bearing either a furanosyl entity or a pyranosyl residue would give a specific signature, even in the gas phase. On the basis of the scope of mass spectrometry, two analytical techniques to register those signatures were considered, i.e. the ion-mobility (IM) and the infra-red multiple photon dissociation (IRMPD), in order to build up cross-linked databases. D-Galactose occurs in natural products in both tautomeric forms and presents all possible regioisomers when linked to D-mannose. Consequently, the four reducing Galf-Manp disaccharides as well as the four Galp-Manp counterparts were firstly synthesized according to a highly convergent approach, and IM-MS and IRMPD-MS data were secondly collected. Both techniques used afforded signatures, specific to the nature of the connectivity between the two glycosyl entities.

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Decoding Protein Gas‐Phase Stability with Alanine Scanning and Collision‐Induced Unfolding Ion Mobility Mass Spectrometry

Analysis & Sensing ◽

10.1002/anse.202000038 ◽

2021 ◽

Author(s):

Jeddidiah Bellamy‐Carter ◽

Louisa O'Grady ◽

Munro Passmore ◽

Matthew Jenner ◽

Neil J. Oldham

Keyword(s):

Mass Spectrometry ◽

Gas Phase ◽

Phase Stability ◽

Ion Mobility ◽

Ion Mobility Mass Spectrometry ◽

Alanine Scanning ◽

Collision Induced Unfolding

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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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Discrimination of patterns of N-acetylation in chitooligosaccharides by gas phase IR spectroscopy integrated to mass spectrometry

Pure and Applied Chemistry ◽

10.1515/pac-2017-0110 ◽

2017 ◽

Vol 89 (9) ◽

pp. 1349-1357 ◽

Cited By ~ 12

Author(s):

Jasper Wattjes ◽

Baptiste Schindler ◽

Stéphane Trombotto ◽

Laurent David ◽

Bruno M. Moerschbacher ◽

...

Keyword(s):

Mass Spectrometry ◽

Ir Spectroscopy ◽

Gas Phase ◽

Dimensional Analysis ◽

Unique Combination ◽

Structural Detail ◽

Chitosan Oligosaccharides ◽

Infra Red ◽

Rapid Discrimination ◽

Gas Phase Spectroscopy

AbstractWe propose a novel, bi-dimensional analysis of partially N-acetylated chitosan oligosaccharides based on gas phase Infra-Red spectroscopy integrated to mass spectrometry (MS). By providing simultaneously MS and IR fingerprints, this approach combines the advantages of MS with the refined structural detail offered by gas phase spectroscopy and provides robust signatures for the rapid discrimination of the patterns of N-acetylation. Four mono-N-deacetylated and two doubly-N-deacetylated chitosan tetramer standards with well-defined patterns of acetylation were produced and analyzed by IR integrated to MS. We show that each sequence displays a unique combination of MS and IR fingerprints, thus offering a rapid diagnostic for the pattern of acetylation without the need for reducing end labeling.

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Effects of Charge State on the Structures of Serum Albumin Ions in the Gas Phase: Insights from Cation-to-Anion Proton-Transfer Reactions, Ion Mobility, and Mass Spectrometry

The Journal of Physical Chemistry B ◽

10.1021/acs.jpcb.8b08427 ◽

2018 ◽

Vol 122 (43) ◽

pp. 9947-9955 ◽

Cited By ~ 4

Author(s):

Meagan M. Gadzuk-Shea ◽

Matthew F. Bush

Keyword(s):

Mass Spectrometry ◽

Proton Transfer ◽

Serum Albumin ◽

Gas Phase ◽

Charge State ◽

Ion Mobility ◽

Transfer Reactions ◽

Proton Transfer Reactions

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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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Native ion mobility mass spectrometry reveals that small organic acid fragments impart gas‐phase stability to carbonic anhydrase II

Rapid Communications in Mass Spectrometry ◽

10.1002/rcm.8570 ◽

2020 ◽

Vol 34 (2) ◽

Author(s):

Clinton G.L. Veale ◽

Maria Mateos Jimenez ◽

C. Logan Mackay ◽

David J. Clarke

Keyword(s):

Mass Spectrometry ◽

Carbonic Anhydrase ◽

Organic Acid ◽

Gas Phase ◽

Phase Stability ◽

Ion Mobility ◽

Carbonic Anhydrase Ii ◽

Ion Mobility Mass Spectrometry

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Deprotonated carbohydrate anion fragmentation chemistry: structural evidence from tandem mass spectrometry, infra-red spectroscopy, and theory

Physical Chemistry Chemical Physics ◽

10.1039/c8cp02620c ◽

2018 ◽

Vol 20 (44) ◽

pp. 27897-27909 ◽

Cited By ~ 7

Author(s):

Jordan M. Rabus ◽

Daniel R. Simmons ◽

Philippe Maître ◽

Benjamin J. Bythell

Keyword(s):

Mass Spectrometry ◽

Infrared Spectroscopy ◽

Tandem Mass Spectrometry ◽

Gas Phase ◽

Tandem Mass ◽

Phase Structures ◽

Infra Red ◽

Fragmentation Chemistry

We investigate the gas-phase structures and fragmentation chemistry of deprotonated carbohydrate anions using combined tandem mass spectrometry, infrared spectroscopy, regioselective labelling, and theory.

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