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

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

Journal of the American Society for Mass Spectrometry ◽

10.1007/s13361-019-02148-z ◽

2019 ◽

Vol 30 (5) ◽

pp. 876-885 ◽

Cited By ~ 8

Author(s):

Hanliu Wang ◽

Joseph Eschweiler ◽

Weidong Cui ◽

Hao Zhang ◽

Carl Frieden ◽

...

Keyword(s):

Mass Spectrometry ◽

Apolipoprotein E ◽

Gas Phase ◽

Electron Capture ◽

Electron Capture Dissociation ◽

Ion Mobility ◽

Structural Information ◽

Native Mass Spectrometry

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

10.26434/chemrxiv.7388723 ◽

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>

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Enhancing top-down proteomics of brain tissue with FAIMS

10.1101/2021.01.18.427216 ◽

2021 ◽

Author(s):

James M. Fulcher ◽

Aman Makaju ◽

Ronald J. Moore ◽

Mowei Zhou ◽

David A. Bennett ◽

...

Keyword(s):

Alzheimer’S Disease ◽

Alzheimer's Disease ◽

Gas Phase ◽

Brain Tissue ◽

Ion Mobility ◽

Asymmetric Waveform ◽

High Complexity ◽

Top Down ◽

Proteome Coverage ◽

The Mean

AbstractProteomic investigations of Alzheimer’s and Parkinson’s disease have provided valuable insights into neurodegenerative disorders. Thus far, these investigations have largely been restricted to bottom-up approaches, hindering the degree to which one can characterize a protein’s “intact” state. Top-down proteomics (TDP) overcomes this limitation, however it is typically limited to observing only the most abundant proteoforms and of a relatively small size. Therefore, offline fractionation techniques are commonly used to reduce sample complexity, limiting throughput. A higher throughput alternative is online fractionation, such as gas phase high-field asymmetric waveform ion mobility spectrometry (FAIMS). Utilizing a high complexity sample derived from Alzheimer’s disease brain tissue, we describe how the addition of FAIMS to TDP can robustly improve the depth of proteome coverage. For example, implementation of FAIMS at −50 compensation voltage (CV) more than doubled the mean number of non-redundant proteoforms observed (1,833 ± 17, n = 3), compared to without (754 ± 35 proteoforms). We also found FAIMS can influence the transmission of proteoforms and their charge envelopes based on their size. Importantly, FAIMS enabled the identification of intact amyloid beta (Aβ) proteoforms, including the aggregation-prone Aβ1-42 variant which is strongly linked to Alzheimer’s disease.

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

Analytical Methods ◽

10.1039/d1ay01461g ◽

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...

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Protein shape sampled by ion mobility mass spectrometry consistently improves protein structure prediction

10.1101/2021.05.27.445812 ◽

2021 ◽

Author(s):

SM Bargeen Alam Turzo ◽

Justin Thomas Seffernick ◽

Amber D Rolland ◽

Micah T Donor ◽

Sten Heinze ◽

...

Keyword(s):

Mass Spectrometry ◽

Protein Structure ◽

Ion Mobility ◽

Structure Determination ◽

Tertiary Structure ◽

De Novo ◽

Structural Information ◽

Collision Cross Section ◽

Protein Structures ◽

Protein Shape

Among a wide variety of mass spectrometry (MS) methodologies available for structural characterizations of proteins, ion mobility (IM) provides structural information about protein shape and size in the form of an orientationally averaged collision cross-section (CCS). While IM data have been predominantly employed for the structural assessment of protein complexes, CCS data from IM experiments have not yet been used to predict tertiary structure from sequence. Here, we are showing that IM data can significantly improve protein structure determination using the modeling suite Rosetta. The Rosetta Projection Approximation using Rough Circular Shapes (PARCS) algorithm was developed that allows for fast and accurate prediction of CCS from structure. Following successful rigorous testing for accuracy, speed, and convergence of PARCS, an integrative modelling approach was developed in Rosetta to use CCS data from IM experiments. Using this method, we predicted protein structures from sequence for a benchmark set of 23 proteins. When using IM data, the predicted structure improved or remained unchanged for all 23 proteins, compared to the predicted models in the absence of CCS data. For 15/23 proteins, the RMSD (root-mean-square deviation) of the predicted model was less than 5.50 Å, compared to only 10/23 without IM data. We also developed a confidence metric that successfully identified near-native models in the absence of a native structure. These results demonstrate the ability of IM data in de novo structure determination.

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Deeper Protein Identification by Using FAIMS in Top-down Proteomics

10.26434/chemrxiv.13653578.v1 ◽

2021 ◽

Author(s):

Robert Gerbasi ◽

Rafael D. Melani ◽

Susan E. Abbatiello ◽

Michael W. Belford ◽

Romain Huguet ◽

...

Keyword(s):

Mass Spectrometry ◽

Molecular Weight ◽

Gas Phase ◽

Ion Mobility ◽

Protein Identification ◽

Signal To Noise ◽

Top Down ◽

Gas Phase Separation ◽

Identification And Characterization

<div> <p>Field Asymmetric Ion Mobility Spectrometry (FAIMS), when used in proteomics studies, provides superior selectivity, and enables more proteins to be identified by providing additional gas phase separation. Here, we tested the performance of cylindrical FAIMS for the identification and characterization of proteoforms by top-down mass spectrometry of heterogeneous protein mixtures. Combining FAIMS with chromatographic separation resulted in a 62% increase in protein identifications and an 8% increase in proteoform identifications as compared to samples analyzed without FAIMS. This increase was attributable, in part, to improved signal-to-noise for proteoforms with similar retention times. Additionally, our results show that the optimal compensation voltage of any given proteoform was correlated with the molecular weight of the analyte. Collectively these results suggest that the addition of FAIMS can enhance top-down proteomics in both discovery and targeted applications. </p> </div>

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Fenton-Chemistry-Based Oxidative Modification of Proteins Reflects Their Conformation

International Journal of Molecular Sciences ◽

10.3390/ijms22189927 ◽

2021 ◽

Vol 22 (18) ◽

pp. 9927

Author(s):

Thomas Nehls ◽

Tim Heymann ◽

Christian Meyners ◽

Felix Hausch ◽

Frederik Lermyte

Keyword(s):

Mass Spectrometry ◽

Protein Structure ◽

Conformational Changes ◽

Structural Information ◽

Mass Measurement ◽

Native Mass Spectrometry ◽

Oxidative Modification ◽

Fenton Chemistry ◽

Analysis Workflow ◽

Complementary Techniques

In order to understand protein structure to a sufficient extent for, e.g., drug discovery, no single technique can provide satisfactory information on both the lowest-energy conformation and on dynamic changes over time (the ‘four-dimensional’ protein structure). Instead, a combination of complementary techniques is required. Mass spectrometry methods have shown promise in addressing protein dynamics, but often rely on the use of high-end commercial or custom instruments. Here, we apply well-established chemistry to conformation-sensitive oxidative protein labelling on a timescale of a few seconds, followed by analysis through a routine protein analysis workflow. For a set of model proteins, we show that site selectivity of labelling can indeed be rationalised in terms of known structural information, and that conformational changes induced by ligand binding are reflected in the modification pattern. In addition to conventional bottom-up analysis, further insights are obtained from intact mass measurement and native mass spectrometry. We believe that this method will provide a valuable and robust addition to the ‘toolbox’ of mass spectrometry researchers studying higher-order protein structure.

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