scholarly journals Effective Discrimination of Gas-Phase Peptide Conformers using TIMS-ECD-ToF MS/MS

2021 ◽  
Author(s):  
Kevin Jeanne dit fouque ◽  
Malte Wellmann ◽  
Dennys Leyva ◽  
Miguel Santos-Fernandez ◽  
Yarixa Cintron Diaz ◽  
...  
Keyword(s):  
Substance P ◽  
Gas Phase ◽  
Ion Mobility ◽  
Structural Information ◽  
Angiotensin I ◽  
Tof Ms ◽  
Model Peptides

In the present work, four, well-studied, model peptides (e.g., substance P, bradykinin, angiotensin I and AT-Hook 3) were used to correlate structural information provided by ion mobility and ECD/CID fragmentation...

scholarly journals Gas Phase Stability of Protein Ions in a Cyclic Ion Mobility Spectrometry Travelling Wave Device

2018 ◽  
Author(s):  
Charles Eldrid ◽  
Jakub Ujma ◽  
Symeon Kalfas ◽  
nick tomczyk ◽  
Kevin Giles ◽  
...  
Keyword(s):  
Cytochrome C ◽  
Gas Phase ◽  
Ion Mobility ◽  
Structural Changes ◽  
Structural Information ◽  
Travelling Wave ◽  
Resolving Power ◽  
Small Proteins ◽  
Multimeric Complex ◽  
Protein Ions

<div>Ion mobility mass spectrometry (IM-MS) allows separation of native protein ions into “conformational families”. Increasing the IM resolving power should allow finer structural information to be obtained, and can be achieved by increasing the length of the IM separator. This, however, increases the time that protein ions spend in the gas phase and previous experiments have shown that the initial conformations of small proteins can be lost within tens of milliseconds. Here, we report on investigations of protein ion stability using a multi-pass travelling wave (TW) cyclic IM (cIM) device. Using this device, minimal structural changes were observed for Cytochrome C after hundreds of milliseconds, while no changes were observed for a larger multimeric complex (Concanavalin A). The geometry of the instrument (Q-cIM-ToF) also enables complex tandem IM experiments to be performed which were used to obtain more detailed collision induced unfolding pathways for Cytochrome C. The novel instrument geometry provide unique capabilities with the potential to expand the field of protein analysis via IM-MS.</div>

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

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.

scholarly journals Gas Phase Stability of Protein Ions in a Cyclic Ion Mobility Spectrometry Travelling Wave Device

2018 ◽  
Author(s):  
Charles Eldrid ◽  
Jakub Ujma ◽  
Symeon Kalfas ◽  
nick tomczyk ◽  
Kevin Giles ◽  
...  
Keyword(s):  
Cytochrome C ◽  
Gas Phase ◽  
Ion Mobility ◽  
Structural Changes ◽  
Structural Information ◽  
Travelling Wave ◽  
Resolving Power ◽  
Small Proteins ◽  
Multimeric Complex ◽  
Protein Ions

<div>Ion mobility mass spectrometry (IM-MS) allows separation of native protein ions into “conformational families”. Increasing the IM resolving power should allow finer structural information to be obtained, and can be achieved by increasing the length of the IM separator. This, however, increases the time that protein ions spend in the gas phase and previous experiments have shown that the initial conformations of small proteins can be lost within tens of milliseconds. Here, we report on investigations of protein ion stability using a multi-pass travelling wave (TW) cyclic IM (cIM) device. Using this device, minimal structural changes were observed for Cytochrome C after hundreds of milliseconds, while no changes were observed for a larger multimeric complex (Concanavalin A). The geometry of the instrument (Q-cIM-ToF) also enables complex tandem IM experiments to be performed which were used to obtain more detailed collision induced unfolding pathways for Cytochrome C. The novel instrument geometry provide unique capabilities with the potential to expand the field of protein analysis via IM-MS.</div>

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 Cyclic Ion Mobility–Collision Activation Experiments Elucidate Protein Behavior in the Gas Phase

2021 ◽  
Author(s):  
Charles Eldrid ◽  
Aisha Ben-Younis ◽  
Jakub Ujma ◽  
Hannah Britt ◽  
Tristan Cragnolini ◽  
...  
Keyword(s):  
Gas Phase ◽  
Ion Mobility ◽  
Collision Activation

scholarly journals Di-Tyrosine Cross-Link Decreases the Collisional Cross-Section of Aβ Peptide Dimers and Trimers in the Gas Phase: An Ion Mobility Study

PLoS ONE ◽  
2014 ◽  
Vol 9 (6) ◽  
pp. e100200 ◽  
Author(s):  
Ewa Sitkiewicz ◽  
Jacek Olędzki ◽  
Jarosław Poznański ◽  
Michał Dadlez
Keyword(s):  
Gas Phase ◽  
Cross Section ◽  
Ion Mobility ◽  
Aβ Peptide ◽  
Cross Link ◽  
Collisional Cross Section ◽  
Mobility Study
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