Proteomic Analysis of a hom-disrupted, Cephamycin C Overproducing Streptomyces clavuligerus

2020 ◽  
Vol 27 ◽  
Author(s):  
Eser Ünsaldı ◽  
Aslıhan Kurt-Kızıldoğan ◽  
Servet Özcan ◽  
Dörte Becher ◽  
Birgit Voigt ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Dna Binding ◽  
Response Regulator ◽  
Streptomyces Clavuligerus ◽  
Homoserine Dehydrogenase ◽  
Cephamycin C ◽  
Flight Mass Spectrometry ◽  
Clusters Of Orthologous Groups ◽  
Proteome Level ◽  
Liquid Chromatography Tandem Mass

Background: Streptomyces clavuligerus is prolific producer of cephamycin C, a medically important antibiotic. In our former study, cephamycin C titer was 2-fold improved by disrupting homoserine dehydrogenase (hom) gene of aspartate pahway in Streptomyces clavuligerus NRRL3585. Objective: In this article, we aimed to provide a comprehensive understanding at the proteome level on potential complex metabolic changes as a consequence of hom disruption in Streptomyces clavuligerus AK39. Methods: A comparative proteomics study was carried out between the wild type and its hom disrupted AK39 strain by 2 Dimensional Electrophoresis-Matrix Assisted Laser Desorption and Ionization Time-Of-Flight Mass Spectrometry (2DE MALDI-TOF/MS) and Nanoscale Liquid Chromatography-Tandem Mass Spectrometry (nanoLC-MS/MS) analyses. Clusters of Orthologous Groups (COG) database was used to determine the functional categories of the proteins. The theoretical pI and Mw values of the proteins were calculated using Expasy pI/Mw tool. Results: “Hypothetical/Unknown” and “Secondary Metabolism” were the most prominent categories of the differentially expressed proteins. Upto 8.7-fold increased level of the positive regulator CcaR was a key finding since CcaR was shown to bind to cefF promoter thereby direcly controlling its expression. Consistently, CeaS2, the first enzyme of CA biosynthetic pathway, was 3.3-fold elevated. There were also many underrepresented proteins associated with the biosynthesis of several Non-Ribosomal Peptide Synthases (NRPSs), clavams, hybrid NRPS/Polyketide synthases (PKSs) and tunicamycin. The most conspicuously underrepresented protein of amino acid metabolism was 4-Hydroxyphenylpyruvate dioxygenase (HppD) acting in tyrosine catabolism. The levels of a Two Component System (TCS) response regulator containing a CheYlike receiver domain and an HTH DNA-binding domain as well as DNA-binding protein HU were elevated while a TetRfamily transcriptional regulator was underexpressed. Conclusion: The results obtained herein will aid in finding out new targets for further improvement of cephamycin C production in Streptomyces clavuligerus.

scholarly journals Metabolic Profiling of Pitaya (Hylocereus polyrhizus) during Fruit Development and Maturation

Molecules ◽  
2019 ◽  
Vol 24 (6) ◽  
pp. 1114 ◽  
Author(s):  
Yawei Wu ◽  
Juan Xu ◽  
Yizhong He ◽  
Meiyan Shi ◽  
Xiumei Han ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Metabolic Profiling ◽  
Developmental Stages ◽  
Principal Component ◽  
Nutrient Content ◽  
Flight Mass Spectrometry ◽  
Potential Biomarker ◽  
Fruit Samples ◽  
Liquid Chromatography Tandem Mass ◽  
Hylocereus Polyrhizus

Pitaya (Hylocereus polyrhizus) has attracted much interest from consumers as it is a novelty fruit with high nutrient content and a tolerance to drought stress. As a group of attractive pigment- and health-promoting natural compounds, betalains represent a visual feature for pitaya fruit quality. However, little information on the correlation between betalains and relevant metabolites exists so far. Currently, color (Commission International del’Eclairage, CIE) parameters, betalain contents, and untargeted metabolic profiling (gas chromatography-time-of-flight-mass spectrometry, GC–MS and liquid chromatography tandem mass spectrometry, LC–MS) have been examined on ‘Zihonglong’ fruits at nine different developmental stages, and the variation character of the metabolite contents was simultaneously investigated between peel and pulp. Furthermore, principal component analysis (PCA) and partial least-squares discriminant analysis (PLS-DA) were used to explore metabolite profiles from the fruit samples. Our results demonstrated that the decrease of amino acid, accompanied by the increase of sugars and organic acid, might contribute to the formation of betalains. Notably, as one of four potential biomarker metabolites, citramalic acid might be related to betalain formation.

scholarly journals Qualitative analysis of 7- and 8-hydroxyzolpidem and discovery of novel zolpidem metabolites in postmortem urine using liquid chromatography–tandem mass spectrometry

2022 ◽  
Author(s):  
Koji Yamaguchi ◽  
Hajime Miyaguchi ◽  
Youkichi Ohno ◽  
Yoshimasa Kanawaku
Keyword(s):  
Mass Spectrometry ◽  
Liquid Chromatography ◽  
Pyridine Ring ◽  
Multiple Reaction Monitoring ◽  
Fragmentation Pattern ◽  
The Novel ◽  
Flight Mass Spectrometry ◽  
Urine Extract ◽  
Liquid Chromatography Tandem Mass ◽  
Characteristic Fragmentation

Abstract Purpose Zolpidem (ZOL) is a hypnotic sometimes used in drug-facilitated crimes. Understanding ZOL metabolism is important for proving ZOL intake. In this study, we synthesized standards of hydroxyzolpidems with a hydroxy group attached to the pyridine ring and analyzed them to prove their presence in postmortem urine. We also searched for novel ZOL metabolites in the urine sample using liquid chromatography–triple quadrupole mass spectrometry (LC-QqQMS) and liquid chromatography–quadrupole time-of-flight mass spectrometry (LC-QqTOFMS). Methods 7- and 8-Hydroxyzolpidem (7OHZ and 8OHZ, respectively) were synthesized and analyzed using LC-QqQMS. Retention times were compared between the synthetic standards and extracts of postmortem urine. To search for novel ZOL metabolites, first, the urine extract was analyzed with data-dependent acquisition, and the peaks showing the characteristic fragmentation pattern of ZOL were selected. Second, product ion spectra of these peaks at various collision energies were acquired and fragments that could be used for multiple reaction monitoring (MRM) were chosen. Finally, MRM parameters were optimized using the urine extract. These peaks were also analyzed using LC-QqTOFMS. Results The presence of 7OHZ and 8OHZ in urine was confirmed. The highest peak among hydroxyzolpidems was assigned to 7OHZ. The novel metabolites found were zolpidem dihydrodiol and its glucuronides, cysteine adducts of ZOL and dihydro(hydroxy)zolpidem, and glucuronides of hydroxyzolpidems. Conclusions The presence of novel metabolites revealed new metabolic pathways, which involve formation of an epoxide on the pyridine ring as an intermediate.

scholarly journals Case Series of Novel Illicit Opioid-Related Deaths

2017 ◽  
Vol 7 (3) ◽  
pp. 477-486 ◽  
Author(s):  
Donna Papsun ◽  
Amy Hawes ◽  
Amanda L.A. Mohr ◽  
Melissa Friscia ◽  
Barry K. Logan
Keyword(s):  
Mass Spectrometry ◽  
Liquid Chromatography ◽  
Whole Blood ◽  
High Performance ◽  
Case Series ◽  
Forensic Toxicology ◽  
Smoke Inhalation ◽  
Manner Of Death ◽  
Flight Mass Spectrometry ◽  
Liquid Chromatography Tandem Mass

Novel illicit opioids, such as furanyl fentanyl and U-47700, are being encountered with increasing frequency in street heroin samples and have been confirmed in a series of overdose deaths in Tennessee. In this paper, we report the pathology and toxicology from 11 deaths involving furanyl fentanyl and U-47700. Routine toxicology was performed on postmortem femoral or antemortem hospital blood samples with targeted broad spectrum drug screening using liquid chromatography-time-of-flight mass spectrometry (LC-TOF/MS). Confirmation and quantitation of the opioid agonists U-47700 and furanyl fentanyl was performed by ultra-high-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) using a novel method. Two cases were identified as containing U-47700 in whole blood (189 and 547 ng/mL), and nine cases contained furanyl fentanyl in whole blood, with concentrations ranging from 2.0 – 42.9 ng/mL. In all 11 cases, the manner of death was deemed accident, with drug intoxication being the primary cause of death; one case was complicated by smoke inhalation. All of the decedents were males ranging from 18-62 years, with the median age being 36 years old. The successful identification and confirmation of these novel illicit opioids in this case series relied on the comprehensive investigation and collaboration of scene investigation, forensic pathology, and forensic toxicology.

Concentrations of para-Fluorofuranylfentanyl (FFF) in Paired Central and Peripheral Blood Collected During Postmortem Death Investigations

2021 ◽  
Author(s):  
Judith Rodriguez Salas ◽  
Alex J Krotulski ◽  
Reta Newman ◽  
Jon R Thogmartin ◽  
Amanda L A Mohr ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Liquid Chromatography ◽  
Peripheral Blood ◽  
The United States ◽  
Blood Concentrations ◽  
Pinellas County ◽  
Flight Mass Spectrometry ◽  
Liquid Chromatography Tandem Mass ◽  
Death Investigation ◽  
Medicolegal Death Investigation

Abstract The opioid epidemic in the United States (U.S.) has been associated with an increasing mortality rate in large part due to the emergence and proliferation of synthetic opioids over the last fifteen years. Fentanyl and its analogues have played a large part in these statistics due to their potency and toxicity. Fluorofuranylfentanyl (FFF) is a fentanyl analogue that emerged in the U.S. in 2018 and was associated with numerous adverse events and deaths. During this study, a liquid chromatography tandem mass spectrometry (LC-MS/MS) workflow was developed to accurately identify the isomer of FFF present (ortho- vs. meta- vs. para-) in medicolegal death investigation cases from Pinellas County, Florida. FFF was quantified in central and peripheral blood samples collected at autopsy. In addition, the metabolism of FFF was studied using liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QTOF-MS). para-FFF was quantitatively confirmed in 29 postmortem cases; no other isomer of FFF was detected. Central blood concentrations ranged between 0.66 and 73 ng/mL (mean = 11±14 ng/mL, median = 10 ng/mL) and peripheral blood concentrations ranged between 0.53 and 23 ng/mL (mean = 5.7±6.4 ng/mL, median = 2.7 ng/mL). Comparison of central to peripheral blood concentrations were evaluated to determine the possibility of postmortem redistribution (PMR). The metabolism of ortho-FFF was studied and found to undergo metabolic processes similar to fentanyl, producing ortho-fluorofuranyl-norfentanyl, fluoro-4-ANPP, and hydroxylated species. The results of this study demonstrate the toxicity of FFF and its implication in medicolegal death investigations. Laboratories must remain aware of new or re-emerging fentanyl analogues, as they pose significant risks to public health and public safety.

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