Simultaneous Evaluation of a Vaccine Component Microheterogeneity and Conformational Integrity Using Native Mass Spectrometry and Limited Charge Reduction

2021 ◽  
Author(s):  
Cedric E. Bobst ◽  
Justin Sperry ◽  
Olga V. Friese ◽  
Igor A. Kaltashov
Keyword(s):  
Mass Spectrometry ◽  
Native Mass Spectrometry ◽  
Charge Reduction ◽  
Vaccine Component ◽  
Simultaneous Evaluation

scholarly journals Imidazole Derivatives Improve Charge Reduction and Stabilization for Native Mass Spectrometry

2019 ◽  
Vol 91 (22) ◽  
pp. 14765-14772 ◽  
Author(s):  
Julia A. Townsend ◽  
James E. Keener ◽  
Zachary M. Miller ◽  
James S. Prell ◽  
Michael T. Marty
Keyword(s):  
Mass Spectrometry ◽  
Native Mass Spectrometry ◽  
Charge Reduction ◽  
Imidazole Derivatives

Charge Reduction of Membrane Proteins in Native Mass Spectrometry Using Alkali Metal Acetate Salts

2019 ◽  
Author(s):  
John T. Petroff II ◽  
Ailing Tong ◽  
Lawrence Chen ◽  
GregoryT. DeKoster ◽  
Farha Khan ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Membrane Proteins ◽  
Alkali Metal ◽  
Conformational Changes ◽  
Alkali Metals ◽  
Native Mass Spectrometry ◽  
Metal Salts ◽  
Charge Reduction ◽  
Alkali Metal Salts ◽  
Metal Acetate

<p>Native mass spectrometry paired with ion mobility (IM-MS) provides the capacity to monitor the structure of protein complexes and simultaneously assess small molecule binding to the protein. Native IM-MS typically utilizes positive mode electrospray ionization producing a distribution of multiply charged protein species. For membrane proteins, these charge states are often too high resulting in protein gas phase unfolding or loss of noncovalent interactions. In an effort to reduce the charge of membrane proteins, the utility of alkali metal salts as a charge reducing additive was explored. Low concentrations of alkali metal salts caused marked charge reduction in the membrane protein, ELIC. The charge reducing effect was only present in membrane proteins, and could not be accounted for by conformational changes in ELIC structure. Charge reduction by alkali metal salts was also detergent dependent, and was most pronounced in long PEG-based detergents such as C10E5 and C12E8. Based on these results, a mechanism was posited for alkali metal charge reduction of membrane proteins. Addition of low concentration of alkali metals may provide an advantageous approach for charge reduction of detergent solubilized membrane proteins by native MS. <br></p>

scholarly journals Charge Reduction of Membrane Proteins in Native Mass Spectrometry Using Alkali Metal Acetate Salts

2020 ◽  
Vol 92 (9) ◽  
pp. 6622-6630 ◽  
Author(s):  
John T. Petroff ◽  
Ailing Tong ◽  
Lawrence J. Chen ◽  
Gregory T. Dekoster ◽  
Farha Khan ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Membrane Proteins ◽  
Alkali Metal ◽  
Native Mass Spectrometry ◽  
Charge Reduction ◽  
Metal Acetate

Charge Reduction of Membrane Proteins in Native Mass Spectrometry Using Alkali Metal Acetate Salts

2019 ◽  
Author(s):  
John T. Petroff II ◽  
Ailing Tong ◽  
Lawrence Chen ◽  
GregoryT. DeKoster ◽  
Farha Khan ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Membrane Proteins ◽  
Alkali Metal ◽  
Conformational Changes ◽  
Alkali Metals ◽  
Native Mass Spectrometry ◽  
Metal Salts ◽  
Charge Reduction ◽  
Alkali Metal Salts ◽  
Metal Acetate

<p>Native mass spectrometry paired with ion mobility (IM-MS) provides the capacity to monitor the structure of protein complexes and simultaneously assess small molecule binding to the protein. Native IM-MS typically utilizes positive mode electrospray ionization producing a distribution of multiply charged protein species. For membrane proteins, these charge states are often too high resulting in protein gas phase unfolding or loss of noncovalent interactions. In an effort to reduce the charge of membrane proteins, the utility of alkali metal salts as a charge reducing additive was explored. Low concentrations of alkali metal salts caused marked charge reduction in the membrane protein, ELIC. The charge reducing effect was only present in membrane proteins, and could not be accounted for by conformational changes in ELIC structure. Charge reduction by alkali metal salts was also detergent dependent, and was most pronounced in long PEG-based detergents such as C10E5 and C12E8. Based on these results, a mechanism was posited for alkali metal charge reduction of membrane proteins. Addition of low concentration of alkali metals may provide an advantageous approach for charge reduction of detergent solubilized membrane proteins by native MS. <br></p>

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.

Sign in / Sign up

Export Citation Format

Share Document