Multiply Charged Cation Attachment to Facilitate Mass Measurement in Negative-Mode Native Mass Spectrometry

Author(s):  
Anthony M. Pitts-McCoy ◽  
Abdirahman M. Abdillahi ◽  
Kenneth W. Lee ◽  
Scott A. McLuckey
2021 ◽  
Vol 22 (18) ◽  
pp. 9927
Author(s):  
Thomas Nehls ◽  
Tim Heymann ◽  
Christian Meyners ◽  
Felix Hausch ◽  
Frederik Lermyte

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.


2019 ◽  
Author(s):  
Zachary VanAernum ◽  
Florian Busch ◽  
Benjamin J. Jones ◽  
Mengxuan Jia ◽  
Zibo Chen ◽  
...  

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.


2020 ◽  
Author(s):  
Paul Dominic B. Olinares ◽  
Jin Young Kang ◽  
Eliza Llewellyn ◽  
Courtney Chiu ◽  
James Chen ◽  
...  

2020 ◽  
Vol 21 ◽  
Author(s):  
Zedong Xiang ◽  
Shaoping Wang ◽  
Haoran Li ◽  
Pingping Dong ◽  
Fan Dong ◽  
...  

Background:: Catalpol, an iridoid glycoside, is one of the richest bioactive components present in Rehmannia glutinosa. More and more metabolites of drugs have exhibit various pharmacological effects, thus providing guidance for clinical application. However, few researches have paid attention on the metabolism of catalpol. Objective:: This study aimed to establish a rapid and effective method to identify catalpol metabolites and evaluate the biotransformation pathways of catalpol in rats. Methods:: In this study, catalpol metabolites in rat urine, plasma and faeces were analyzed by UHPLC-Q-Exactive MS for the characterization of metabolism of catalpol. Based on high-resolution extracted ion chromatograms (HREICs) and parallel reaction monitoring mode (PRM), metabolites of catalpol were identified by comparing the diagnostic product ions (DPIs), chromatographic retention times, neutral loss fragments (NLFs) and accurate mass measurement with those of catalpol reference standard. Results: A total of 29 catalpol metabolites were detected and identified in both negative and positive ion modes. Nine metabolic reactions including deglycosylation, hydroxylation, dihydroxylation, hydrogenation, dehydrogenation, oxidation of methylene to ketone, glucuronidation, glycine conjugation and cysteine conjugation were proposed. Conclusion:: A rapid and effective method based on UHPLC-Q-Exactive MS was developed to mine the metabolism information of catalpol. Results of metabolites and biotransformation pathways of catalpol suggested that when orally administrated, catalpol was firstly metabolized into catalpol aglycone, after which phase Ⅰ and phase Ⅱ reactions occurred. However, hydrophilic chromatography-mass spectrometry still needed to further find the polar metabolites of catalpol.


2021 ◽  
Author(s):  
Anirban Ghosh ◽  
Eric Largy ◽  
Valérie Gabelica

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).


2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Miaomiao Liu ◽  
Wesley C. Van Voorhis ◽  
Ronald J. Quinn

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