scholarly journals Intrinsic GTPase Activity of K-RAS Monitored by Native Mass Spectrometry

Biochemistry ◽  
2019 ◽  
Vol 58 (31) ◽  
pp. 3396-3405 ◽  
Author(s):  
Zahra Moghadamchargari ◽  
Jamison Huddleston ◽  
Mehdi Shirzadeh ◽  
Xueyun Zheng ◽  
David E. Clemmer ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Native Mass Spectrometry ◽  
Gtpase Activity ◽  
Intrinsic Gtpase Activity

Rapid Online Buffer Exchange: A Method for Screening of Proteins, Protein Complexes, and Cell Lysates by Native Mass Spectrometry

2019 ◽  
Author(s):  
Zachary VanAernum ◽  
Florian Busch ◽  
Benjamin J. Jones ◽  
Mengxuan Jia ◽  
Zibo Chen ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
High Speed ◽  
Structural Information ◽  
Protein Complexes ◽  
High Sensitivity ◽  
Native Mass Spectrometry ◽  
Structural Features ◽  
Consumer Products ◽  
Cell Lysates ◽  
Protein Expression And Purification

It is important to assess the identity and purity of proteins and protein complexes during and after protein purification to ensure that samples are of sufficient quality for further biochemical and structural characterization, as well as for use in consumer products, chemical processes, and therapeutics. Native mass spectrometry (nMS) has become an important tool in protein analysis due to its ability to retain non-covalent interactions during measurements, making it possible to obtain protein structural information with high sensitivity and at high speed. Interferences from the presence of non-volatiles are typically alleviated by offline buffer exchange, which is timeconsuming and difficult to automate. We provide a protocol for rapid online buffer exchange (OBE) nMS to directly screen structural features of pre-purified proteins, protein complexes, or clarified cell lysates. Information obtained by OBE nMS can be used for fast (<5 min) quality control and can further guide protein expression and purification optimization.

Native Mass Spectrometry-Based Screening for Optimal Sample Preparation in Single Particle Cryo-EM

2020 ◽  
Author(s):  
Paul Dominic B. Olinares ◽  
Jin Young Kang ◽  
Eliza Llewellyn ◽  
Courtney Chiu ◽  
James Chen ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Sample Preparation ◽  
Single Particle ◽  
Native Mass Spectrometry ◽  
Optimal Sample

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 Development of a target identification approach using native mass spectrometry

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Miaomiao Liu ◽  
Wesley C. Van Voorhis ◽  
Ronald J. Quinn
Keyword(s):  
Mass Spectrometry ◽  
Protein Interactions ◽  
Target Identification ◽  
Native Mass Spectrometry ◽  
Pharmaceutical Drugs ◽  
Ligand Complex ◽  
Experimental Conditions ◽  
Protein Mixture ◽  
Native Ms ◽  
Drug Target Identification

AbstractA key step in the development of new pharmaceutical drugs is the identification of the molecular target and distinguishing this from all other gene products that respond indirectly to the drug. Target identification remains a crucial process and a current bottleneck for advancing hits through the discovery pipeline. Here we report a method, that takes advantage of the specific detection of protein–ligand complexes by native mass spectrometry (MS) to probe the protein partner of a ligand in an untargeted method. The key advantage is that it uses unmodified small molecules for binding and, thereby, it does not require labelled ligands and is not limited by the chemistry required to tag the molecule. We demonstrate the use of native MS to identify known ligand–protein interactions in a protein mixture under various experimental conditions. A protein–ligand complex was successfully detected between parthenolide and thioredoxin (PfTrx) in a five-protein mixture, as well as when parthenolide was mixed in a bacterial cell lysate spiked with PfTrx. We provide preliminary data that native MS could be used to identify binding targets for any small molecule.

Linear epitope mapping by native mass spectrometry

2009 ◽  
Vol 395 (1) ◽  
pp. 100-107 ◽  
Author(s):  
Xiaojun Lu ◽  
Michael R. DeFelippis ◽  
Lihua Huang
Keyword(s):  
Mass Spectrometry ◽  
Epitope Mapping ◽  
Native Mass Spectrometry ◽  
Linear Epitope

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.

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