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1.
Anal Bioanal Chem ; 413(30): 7559-7585, 2021 Dec.
Article in English | MEDLINE | ID: covidwho-1503906

ABSTRACT

Subunit vaccines based on the receptor-binding domain (RBD) of the spike protein of SARS-CoV-2 provide one of the most promising strategies to fight the COVID-19 pandemic. The detailed characterization of the protein primary structure by mass spectrometry (MS) is mandatory, as described in ICHQ6B guidelines. In this work, several recombinant RBD proteins produced in five expression systems were characterized using a non-conventional protocol known as in-solution buffer-free digestion (BFD). In a single ESI-MS spectrum, BFD allowed very high sequence coverage (≥ 99%) and the detection of highly hydrophilic regions, including very short and hydrophilic peptides (2-8 amino acids), and the His6-tagged C-terminal peptide carrying several post-translational modifications at Cys538 such as cysteinylation, homocysteinylation, glutathionylation, truncated glutathionylation, and cyanylation, among others. The analysis using the conventional digestion protocol allowed lower sequence coverage (80-90%) and did not detect peptides carrying most of the above-mentioned PTMs. The two C-terminal peptides of a dimer [RBD(319-541)-(His)6]2 linked by an intermolecular disulfide bond (Cys538-Cys538) with twelve histidine residues were only detected by BFD. This protocol allows the detection of the four disulfide bonds present in the native RBD, low-abundance scrambling variants, free cysteine residues, O-glycoforms, and incomplete processing of the N-terminal end, if present. Artifacts generated by the in-solution BFD protocol were also characterized. BFD can be easily implemented; it has been applied to the characterization of the active pharmaceutical ingredient of two RBD-based vaccines, and we foresee that it can be also helpful to the characterization of mutated RBDs.


Subject(s)
Cysteine/metabolism , Peptide Fragments/metabolism , Protein Processing, Post-Translational , Spectrometry, Mass, Electrospray Ionization/methods , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/metabolism , Amino Acid Sequence , Cysteine/chemistry , Humans , Hydrophobic and Hydrophilic Interactions , Peptide Fragments/chemistry , Protein Binding , Protein Domains , Protein Subunits
2.
Anal Bioanal Chem ; 413(29): 7205-7214, 2021 Dec.
Article in English | MEDLINE | ID: covidwho-1356000

ABSTRACT

Native mass spectrometry (MS) enjoyed tremendous success in the past two decades in a wide range of studies aiming at understanding the molecular mechanisms of physiological processes underlying a variety of pathologies and accelerating the drug discovery process. However, the success record of native MS has been surprisingly modest with respect to the most recent challenge facing the biomedical community-the novel coronavirus infection (COVID-19). The major reason for the paucity of successful studies that use native MS to target various aspects of SARS-CoV-2 interaction with its host is the extreme degree of heterogeneity of the viral protein playing a key role in the host cell invasion. Indeed, the SARS-CoV-2 spike protein (S-protein) is extensively glycosylated, presenting a formidable challenge for native MS as a means of characterizing its interactions with both the host cell-surface receptor ACE2 and the drug candidates capable of disrupting this interaction. In this work, we evaluate the utility of native MS complemented with the experimental methods using gas-phase chemistry (limited charge reduction) to obtain meaningful information on the association of the S1 domain of the S-protein with the ACE2 ectodomain, and the influence of a small synthetic heparinoid on this interaction. Native MS reveals the presence of several different S1 oligomers in solution and allows the stoichiometry of the most prominent S1/ACE2 complexes to be determined. This enables meaningful interpretation of the changes in native MS that are observed upon addition of a small synthetic heparinoid (the pentasaccharide fondaparinux) to the S1/ACE2 solution, confirming that the small polyanion destabilizes the protein/receptor binding.


Subject(s)
Receptors, Virus/metabolism , Spectrometry, Mass, Electrospray Ionization/methods , Spike Glycoprotein, Coronavirus/metabolism , Dimerization , Humans , Protein Binding
3.
J Nat Prod ; 83(12): 3493-3501, 2020 12 24.
Article in English | MEDLINE | ID: covidwho-1351918

ABSTRACT

Svalbardines A and B (1 and 2) and annularin K (3) were isolated from cultures of Poaceicola sp. E1PB, an endophyte isolated from the petals of Papaver dahlianum from Svalbard, Norway. Svalbardine A (1) is a pyrano[3,2-c]chromen-4-one, a new analogue of citromycetin. Svalbardine B (2) displays an unprecedented carbon skeleton based on a 5'-benzyl-spiro[chroman-3,7'-isochromene]-4,8'-dione core. Annularin K (3) is a hydroxylated derivative of annularin D. The structure of these new polyketides, along with those of known compounds 4-6, was established by spectrometric analysis, including extensive ESI-CID-MSn processing in the case of svalbardine B (2).


Subject(s)
Ascomycota/chemistry , Spectrometry, Mass, Electrospray Ionization/methods , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/isolation & purification , Anti-Bacterial Agents/pharmacology , Arctic Regions , Microbial Sensitivity Tests , Molecular Structure , Spectrum Analysis
4.
J Chromatogr B Analyt Technol Biomed Life Sci ; 1176: 122768, 2021 Jun 30.
Article in English | MEDLINE | ID: covidwho-1240421

ABSTRACT

Favipiravir is a broad-spectrum inhibitor of viral RNA polymerase. It is currently used as a possible treatment for coronavirus disease 2019 (COVID-19). Pre-clinical or clinical trials of favipiravir require robust, sensitive, and accurate bioanalytical methods for quantitation of favipiravir levels. Recently, several studies have been reported about developing a validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for measuring favipiravir levels. However, these methods were validated predominantly for plasma samples, electrospray ionization was operated only in negative or positive mode, and clinical application of these methods has not been applied for patients with COVID-19. This study aimed was to develop a validated LC-MS/MS method for the measurement of favipiravir levels in positive and negative electrospray ionization mode and to perform a pilot study in patients with COVID-19 receiving favipiravir to demonstrate the applicability of this method in biological samples. Simple protein precipitation was used for the extraction of favipiravir from the desired matrix. Favipiravir levels were quantitated using MS / MS with an electrospray ionization source in positive and negative multiple reaction monitoring (MRM) mode. The chromatographic detection was performed on a reverse-phase Phenomenex C18 column (50 mm × 4.6 mm, 5 µm, 100 Å) with gradient elution using 0.1% formic acid in water and 0.1% formic acid in methanol as mobile phase. The method was linear over the concentration ranges of 0.048-50 µg/mL (in negative ionization mode) and 0.062-50 µg/mL (in positive ionization mode) with a correlation coefficient (r2) better than 0.998. The total run time was 3.5 min. The intra-assay and inter-assay %CV values were less than 7.2% and 8.0%, respectively. A simple, rapid and robust LC-MS / MS method was developed for the measurement of favipiravir and validation studies were performed. The validated method was successfully applied for drug level measurement in COVID-19 patients receiving favipiravir.


Subject(s)
Amides/blood , COVID-19/drug therapy , Chromatography, Liquid/methods , Pyrazines/blood , Tandem Mass Spectrometry/methods , Amides/administration & dosage , Amides/therapeutic use , Antiviral Agents/administration & dosage , Antiviral Agents/blood , Antiviral Agents/therapeutic use , COVID-19/blood , Drug Stability , Humans , Limit of Detection , Pyrazines/administration & dosage , Pyrazines/therapeutic use , Sensitivity and Specificity , Spectrometry, Mass, Electrospray Ionization/methods
5.
J Pharm Biomed Anal ; 197: 113956, 2021 Apr 15.
Article in English | MEDLINE | ID: covidwho-1078045

ABSTRACT

The clinically tested KCa3.1 channel blocker, senicapoc, has been proven to have excellent pharmacological properties and prior clinical trials found it to be safe for use in patients with sickle cell anaemia. Currently, several preclinical projects are aiming to repurpose senicapoc for other indications, but well-described analytical methods in the literature are lacking. Our aim was to develop a sensitive, rapid and accurate ultra-high-performance liquid chromatography-tandem mass spectrometry method using pneumatically assisted electrospray ionisation (UHPLC-ESI-MS/MS) suitable for the determination of senicapoc in plasma samples. Unfortunately, direct analysis of senicapoc in crude acetonitrile extracts of human plasma samples by UHPLC-ESI-MS/MS was subjected to significant and variable ion suppression from coeluting phospholipids (PLs). The interferences were mainly caused by the presence of phosphatidylcholine and phosphatidylethanolamine classes of PLs, including their lyso-products. However, the PLs were easily removed from crude extracts by filtration through a sorbent with Lewis acid properties which decreased the total ion suppression effect to approximately 5%. Based on this technique, a simple high-throughput UHPLC-MS/MS method was developed and validated for the determination of senicapoc in 100-µL plasma samples. The lower limit of quantification was 0.1 ng/mL. The mean true extraction recovery was close to 100 %. The relative intra-laboratory reproducibility standard deviations of the measured concentrations were 8% and 4% at concentrations of 0.1 ng/mL and 250 ng/mL, respectively. The trueness expressed as the relative bias of the test results was within ± 2% at concentrations of 1 ng/mL or higher.


Subject(s)
Acetamides/blood , Chromatography, High Pressure Liquid/methods , Plasma/chemistry , Tandem Mass Spectrometry/methods , Trityl Compounds/blood , Animals , Female , Filtration/methods , Humans , Limit of Detection , Phospholipids/blood , Reproducibility of Results , Spectrometry, Mass, Electrospray Ionization/methods , Swine
6.
Anal Chem ; 93(5): 2767-2775, 2021 02 09.
Article in English | MEDLINE | ID: covidwho-1039622

ABSTRACT

Clinical tissue specimens are often unscreened, and preparation of tissue sections for analysis by mass spectrometry imaging (MSI) can cause aerosolization of particles potentially carrying an infectious load. We here present a decontamination approach based on ultraviolet-C (UV-C) light to inactivate clinically relevant pathogens such as herpesviridae, papovaviridae human immunodeficiency virus, or SARS-CoV-2, which may be present in human tissue samples while preserving the biodistributions of analytes within the tissue. High doses of UV-C required for high-level disinfection were found to cause oxidation and photodegradation of endogenous species. Lower UV-C doses maintaining inactivation of clinically relevant pathogens to a level of increased operator safety were found to be less destructive to the tissue metabolome and xenobiotics. These doses caused less alterations of the tissue metabolome and allowed elucidation of the biodistribution of the endogenous metabolites. Additionally, we were able to determine the spatially integrated abundances of the ATR inhibitor ceralasertib from decontaminated human biopsies using desorption electrospray ionization-MSI (DESI-MSI).


Subject(s)
Decontamination/methods , Ultraviolet Rays , Animals , Azetidines/analysis , Azetidines/therapeutic use , COVID-19/pathology , COVID-19/virology , Head and Neck Neoplasms/chemistry , Head and Neck Neoplasms/drug therapy , Head and Neck Neoplasms/pathology , Humans , Male , Metabolome , Naphthalenes/analysis , Naphthalenes/therapeutic use , Photolysis/radiation effects , Rats , Rats, Wistar , SARS-CoV-2/isolation & purification , SARS-CoV-2/radiation effects , Spectrometry, Mass, Electrospray Ionization/methods , Terfenadine/chemistry , Virus Inactivation/radiation effects
7.
Food Addit Contam Part A Chem Anal Control Expo Risk Assess ; 38(1): 148-159, 2021 Jan.
Article in English | MEDLINE | ID: covidwho-1024072

ABSTRACT

Coix seed is an important food and traditional Chinese medicine in China and other Asian countries. Notably, coix seed is currently being used as a traditional medicine for the treatment of COVID-19 in China. However, coix seeds are generally contaminated by mycotoxins, and this risk cannot be ignored. In this paper, we developed a method that involves direct extraction and UHPLC-HRMS analysis for the simultaneous detection of 24 mycotoxins in coix seeds. UHPLC-HRMS instrument and data acquisition parameters, and the sample pretreatment were optimised. One-step extraction showed several advantages compared to the three commercial solid-phase extraction clean-up methods, including ease of use, reduced time of sample preparation, low cost, good recovery, and acceptable matrix effect. The method validation results indicate that all mycotoxins have good linearity and sensitivity. Recoveries were between 74.2-101.1%, and RSD ranged from 0.1-5.8%. The LOQs for 24 mycotoxins were in the range of 0.5-100 µg/kg. To survey the contamination levels of these mycotoxins in commercial coix seeds, more than 70 samples were collected from Chinese markets and were analysed using the newly developed method. Zearalenone (positive ratio: 98.7%, range:1.1-1562 µg/kg), deoxynivalenol (positive ratio: 87%, range: 8.4-382.5 µg/kg), nivalenol (positive ratio: 85.7%, range: 26.8-828.2 µg/kg), fumonisin B1 (positive ratio: 84.4%, range:2.5-314.5 µg/kg), fumonisin B2 (positive ratio: 75.3%, range:1.6-72.8 µg/kg), fumonisin B3 (positive ratio: 48%, range:1.0-203.6 µg/kg), aflatoxin B1 (positive ratio: 29.9%, range: 0.39-14.7 µg/kg), sterigmatocystin (positive ratio: 29.9%, range: 1.4-51.6 µg/kg), and tenuazonic acid (positive ratio: 19.5%, range 36.1-105.7 µg/kg) were the most frequent mycotoxin contaminants. These results highlight the importance of routine monitoring and control of mycotoxins in coix seeds.


Subject(s)
COVID-19/drug therapy , Coix/chemistry , Drug Contamination , Drugs, Chinese Herbal/chemistry , Drugs, Chinese Herbal/toxicity , Mycotoxins/analysis , Mycotoxins/toxicity , Antiviral Agents/chemistry , Antiviral Agents/therapeutic use , Antiviral Agents/toxicity , China , Chromatography, High Pressure Liquid/methods , Drugs, Chinese Herbal/therapeutic use , Humans , Pandemics , Risk Assessment , SARS-CoV-2 , Seeds/chemistry , Solvents , Spectrometry, Mass, Electrospray Ionization/methods
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