We present a comparison of three different electrospray-based ionization techniques for the investigation of noncovalent complexes with mass spectrometry. The features and characteristics of standard electrospray ionization (ESI), chip-based nanoESI, and electrosonic spray ionization (ESSI) mounted onto a hybrid quadrupole time-of-flight mass spectrometer were compared in their performance to determine the dissociation constant (K D ) of the model system hen egg white lysozyme (HEWL) binding to N, N N=,
In the present report, a method based on chip-based nanoelectrospray mass spectrometry (nanoESI-MS) is described to detect noncovalent ligand binding to the human estrogen receptor a ligand-binding domain (hERa LBD). This system represents an important environmental interest, because a wide variety of molecules, known as endocrine disruptors, can bind to the estrogen receptor (ER) and induce adverse health effects in wildlife and humans. Using proper experimental conditions, the nanoESI-MS approach allowed for the detection of specific ligand interactions with hERa LBD. The relative gasphase stability of selected hERa LBD-ligand complexes did not mirror the binding affinity in solution, a result that demonstrates the prominent role of hydrophobic contacts for stabilizing ER-ligand complexes in solution. The best approach to evaluate relative solution-binding affinity by nanoESI-MS was to perform competitive binding experiments with 17b-estradiol (E2) used as a reference ligand. Among the ligands tested, the relative binding affinity for hERa LBD measured by nanoESI-MS was 4-hydroxtamoxifen % diethylstilbestrol > E2 >> genistein >> bisphenol A, consistent with the order of the binding affinities in solution. The limited reproducibility of the bound to free protein ratio measured by nanoESI-MS for this system only allowed the binding constants (K d ) to be estimated (low nanomolar range for E2). The specificity of nanoESI-MS combined with its speed (1 min/ligand), low sample consumption (90 pmol protein/ligand), and its sensitivity for ligand (30 ng/mL) demonstrates that this technique is a promising method for screening suspected endocrine disrupting compounds and to qualitatively evaluate their binding affinity.Keywords: electrospray ionization mass spectrometry; noncovalent; nuclear receptor; estrogen receptor; endocrine disruptors; solution affinityThe estrogen receptor (ER) belongs to the nuclear receptor (NR) superfamily of ligand-activated transcription regulators, which are involved in many processes such as growth, organ differentiation, and development of reproductive tissues. The ER, which comprises a DNA-binding domain (DBD) and a ligand-binding domain (LBD), activates the transcription of target genes in response to the binding of estrogens, a group of steroid compounds, to the LBD. Once bound by hormones, the ER undergoes a conformational change that facilitates dimerization and subsequent interactions with the specific DNA sequence (Kumar and Chambon 1988;Steinmetz et al. 2001). The large hydrophobic cavity of the ER LBD allows the binding of a wide variety of nonsteroidal compounds through hydrophobic interactions. In the last decades, a variety of Reprint requests to: Renato Zenobi, Department of Chemistry and Applied Biosciences, ETH Zurich, 8093 Zurich, Switzerland; e-mail: zenobi@org.chem.ethz.ch; fax: 41 44 632 12 92.Article published online ahead of print. Article and publication date are at
BackgroundIdiopathic pulmonary fibrosis (IPF) is a lethal lung disease of unknown etiology. Patients present loss of lung function, dyspnea and dry cough. Diagnosis requires compatible radiologic imaging and, in undetermined cases, invasive procedures such as bronchoscopy and surgical lung biopsy. The pathophysiological mechanisms of IPF are not completely understood. Lung injury with abnormal alveolar epithelial repair is thought to be a major cause for activation of profibrotic pathways in IPF. Metabolic signatures might indicate pathological pathways involved in disease development and progression. Reliable serum biomarker would help to improve both diagnostic approach and monitoring of drug effects.MethodThe global metabolic profiles measured by ultra high-performance liquid chromatography coupled to high-resolution mass spectrometry (UHPLC-HRMS) of ten stable IPF patients were compared to the ones of ten healthy participants. The results were validated in an additional study of eleven IPF patients and ten healthy controls.ResultsWe discovered 10 discriminative metabolic features using multivariate and univariate statistical analysis. Among them, we identified one metabolite at a retention time of 9.59 min that was two times more abundant in the serum of IPF patients compared to healthy participants. Based on its ion pattern, a lysophosphatidylcholine (LysoPC) was proposed. LysoPC is a precursor of lysophosphatidic acid (LPA) – a known mediator for lung fibrosis with its pathway currently being evaluated as new therapeutic drug target for IPF and other fibrotic diseases.ConclusionsWe identified a LysoPC by UHPLC-HRMS as potential biomarker in serum of patients with IPF. Further validation studies in a larger cohort are necessary to determine its role in IPF.Trial RegistrationSerum samples from IPF patients have been obtained within the clinical trial NCT02173145 at baseline and from the idiopathic interstitial pneumonia (IIP) cohort study. The study was approved by the Swiss Ethics Committee, Bern (KEK 002/14 and 246/15 or PB_2016–01524).Electronic supplementary materialThe online version of this article (doi: 10.1186/s12931-018-0714-2) contains supplementary material, which is available to authorized users.
Summary A universal anti‐Xa assay for the determination of rivaroxaban, apixaban and edoxaban drug concentrations would simplify laboratory procedures and facilitate widespread implementation. Following two pilot studies analysing spiked samples and material from 698 patients, we conducted a prospective multicentre cross‐sectional study, including 867 patients treated with rivaroxaban, apixaban or edoxaban in clinical practice to comprehensively evaluate a simple, readily available anti‐Xa assay that would accurately measure drug concentrations and correctly predict relevant levels in clinical practice. Anti‐Xa activity was measured by an assay calibrated with low‐molecular‐weight heparin (LMWH) in addition to ultra‐high performance liquid chromatography‐tandem mass spectrometry (LC‐MS/MS). As an external validation, LMWH‐calibrated anti‐Xa activity was also determined in nine external laboratories. The LMWH‐calibrated anti‐Xa activity correlated strongly with rivaroxaban, apixaban or edoxaban drug levels [rs = 0·98, 95% confidence interval (CI) 0·98–0·98]. The sensitivity for the clinically relevant cut‐off levels of 30, 50 and 100 µg/l was 96·2% (95% CI 94·4–97·4), 96·4% (95% CI 94·4–97·7) and 96·7% (95% CI 94·3–98·1) respectively. Concordant results were obtained in the external validation study. In conclusion, a universal, LMWH‐calibrated anti‐Xa assay accurately measured rivaroxaban, apixaban and edoxaban concentrations and correctly predicted relevant drug concentrations in clinical practice.
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