Aufreinigung und Charakterisierung von Angiotensin-II-generierenden Enzymen aus dem Schweinenierengewebe

Titelblatt und Inhaltsverzeichnis Introduction Methods Results Discussion Summary Literature List of figures and tables List of abbreviations Acknowledgement ErklärungThe object of this work was the purification, characterization and identification of angiotensin-II-generating enzymes from porcine renal tissue. Three different angiotensin-II-generating enzymes were purified using protein liquid chromatography techniques. During the course of purification angiotensin-II-generating enzyme activity was measured and characterized using a novel mass spectrometry based assay system (MES-assay system) for the detection of proteolytic enzyme activities. One active fraction, here named Fraction I was purified using a combination of anion exchange, hydroxyapatite, lectin affinity and chymostatin-antipain-inhibitor affinity chromatography. Protein identification experiments using peptide mass fingerprint identified this enzyme as Cathepsin G. Cathepsin G was detected in kidney for the first time. The second active fraction, here named Fraction II was purified using a combination of anion exchange, hydroxyapatite, and lentil lectin sugar affinity and size exclusion chromatography. Protein identification experiments identified this enzyme as Angiotensin converting enzyme. The successful purification of ACE is a proof of principle for the chosen purification strategy and the novel MES-assay system. The third angiotensin-II-generating enzyme fraction, here named Fraction III was purified using a combination of anion exchange, wheat germ lectin sugar affinity and aprotinin-inhibitor affinity chromatography. The identification of this enzyme did not succeed. Experiments in order to characterize this enzyme fraction were carried out. The molecular weight of the enzyme fraction was approximately 160 kDa. A unique feature of Fraction III was its substrate specifity. Besides its angiotensin-II-generating activity it cleaved the L-kininogen analogue Lys- Bradykinin-Ser-Val-Gln-Val-Ser to form Des-Arg10-Kallidin. The proteolytic activity of Fraction III does not coincide with the known proteases of the Kinintensin system. Further research efforts should be made to reveal the identity of this enzyme.Das Thema dieser Arbeit war die Reinigung, Charakterisierung und Identifizierung von Angiotensin- II generierenden Enzymen aus der Schweineniere. Drei verschiedene Angiotensin- II generierende Enzyme wurden mit Hilfe der Flüssigchromatographie aufgereinigt. Die Angiotensin-II generierende Enzymaktiviät wurde während der Aufreinigung mittels eines neuen Massenspekrometrie basierten Nachweissystems (MES-System) detektiert und charakterisiert. Eine aktive Fraktion, hier Fraktion I genannt, wurde mit einer Kombination von Anionenaustausch-, Hydroxyapatit-, Lectin- und Chymostatin-Antipain-Affinitätschromatographie gereinigt. Mit Hilfe von Proteinidentifizierungsexperimenten wurde das Angiotensin-II generierende Enzym dieser Fraktion als Cathepsin G identifiziert. Cathepsin G wurde das erste Mal in der Niere nachgewiesen. Eine zweite aktive Fraktion, hier Fraktion II genannt, wurde mit einer Kombination von Anionenaustausch-, Hydroxyapatit-, Lectin- und Größenausschlusschromatographie gereinigt. Mit Hilfe von Proteinidenfizierungsexperimenten wurde das Angiotensin-II generierende Enzym dieser Fraktion als Angiotensin Converting Enzym identifiziert. Die erfolgreiche Aufreinigung ein Beweis für das Funktionieren der gewählten Aufreinigungsstrategie und des neuartigen MES- Nachweissystems. Eine dritte aktive Fraktion, hier Fraktion III genannt, wurde mittel einer Kombination aus Anionenaustausch-, Lectin- und Aprotinin- Affinitätschromatographie aufgereinigt. Die Identifizierung des Angiotensin-II generierenden Enzyms dieser Fraktion gelang nicht. Experimente zur Charakterisierung dieser Enzymfraktion wurden durchgeführt. Das Molekulargewicht betrug ca. 160 kDa. Ein besonderes Merkmal dieser Enzymfraktion war die Subtratspezifität. Neben der Angiotensin-II generierende Aktivität spaltete es das L-Kininogen Analogon Lys-Bradikin-Ser-Val-Gln-Ser Substrat proteolytisch zu Des-Arg10-Kallidin. Weitere Anstrengungen müssen unternommen werden, um die Identität dieses Enzyms zu klären

Validation of an automated extraction procedure for amino acids and acylcarnitines for use with tandem mass spectrometry for newborn screening

3 Tablas.- 1 Figura.- Datos suplementarios disponibles en la página web del editor.A certified reagent kit for newborn screening was transferred on a fully automated dried blood spot platform. The dried blood spot cards are directly eluted and the extract is online guided to tandem mass spectrometry instrument, where the amino acid and acyl carnitine panel is detected. The method takes 2 minutes per sample and requires no human interaction for up to 500 samples. The method is fully standardized through the automation and the usage of only certified consumables and reference material. The manual reagent kit was first modified to fit the automated platform, secondly validated and third, successfully transferred into a routine newborn screening laboratory.Peer reviewe

Fully automated drug screening of dried blood spots using online LC-MS/MS analysis

A new and fully automated workflow for the cost effective drug screening of large populations based on the dried blood spot (DBS) technology was introduced in this study. DBS were prepared by spotting 15 μL of whole blood, previously spiked with alprazolam, amphetamine, cocaine, codeine, diazepam, fentanyl, lysergic acid diethylamide (LSD), 3,4-methylenedioxymethamphet-amine (MDMA), methadone, methamphetamine, morphine and oxycodone onto filter paper cards. The dried spots were scanned, spiked with deuterated standards and directly extracted. The extract was transferred online to an analytical LC column and then to the electrospray ionization tandem mass spectrometry system. All drugs were quantified at their cut-off level and good precision and correlation within the calibration range was obtained. The method was finally applied to DBS samples from two patients with back pain and codeine and oxycodone could be identified and quantified accurately below the level of misuse of 89.6 ng/mL and 39.6 ng/mL respectively

Fully automated drug screening of dried blood spots using online LC-MS/MS analysis

A new and fully automated workflow for the cost effective drug screening of large populations based on the dried blood spot (DBS) technology was introduced in this study. DBS were prepared by spotting 15 μL of whole blood, previously spiked with alprazolam, amphetamine, cocaine, codeine, diazepam, fentanyl, lysergic acid diethylamide (LSD), 3,4-methylenedioxymethamphet-amine (MDMA), methadone, methamphetamine, morphine and oxycodone onto filter paper cards. The dried spots were scanned, spiked with deuterated standards and directly extracted. The extract was transferred online to an analytical LC column and then to the electrospray ionization tandem mass spectrometry system. All drugs were quantified at their cut-off level and good precision and correlation within the calibration range was obtained. The method was finally applied to DBS samples from two patients with back pain and codeine and oxycodone could be identified and quantified accurately below the level of misuse of 89.6 ng/mL and 39.6 ng/mL respectively

Automated drug screening of dried blood spots using online LC-MS/MS analysis

2 figuras, 2 tablasA fully automated method for the effective drug screening of large populations based on dried blood spot (DBS) technology is presented. DBSs were prepared, scanned, then spiked with deuterated standards, and directly extracted, before they were transferred online to an analytical liquid chromatography (LC) column and then to the electrospray ionization tandem mass spectrometry (ESI-MS/MS) system. The method was applied to DBS samples from two patients with back pain; codeine and oxycodone could be identified and quantified accurately below the level of misuse of 89.6 ng/mL and 39.6 ng/mL, respectively

Extended and Fully Automated Newborn Screening Method for Mass Spectrometry Detection

A new and fully automated newborn screening method for mass spectrometry was introduced in this paper. Pathological relevant amino acids, acylcarnitines, and certain steroids are detected within 4 min per sample. Each sample is treated in an automated and standardized workflow, where a mixture of deuterated internal standards is sprayed onto the sample before extraction. All compounds showed good linearity, and intra- and inter-day variation lies within the acceptance criteria (except for aspartic acid). The described workflow decreases analysis cost and labor while improving the sample traceability towards good laboratory practice

Mass spectrometry-assisted protease substrate screening

Since sequencing of the human genome was completed, more than 500 genes have been annotated as proteases. Exploring the physiological role of each protease requires the identification of their natural substrates. However, the endogenous substrates of many of the human proteases are as yet unknown. Here we describe a new assay that addresses this problem. The assay, which easily can be automated, is based on the incubation of immobilized protein fractions, which may contain the natural substrate, with a defined protease. After concentrating the proteolytically released peptides by reversed-phase chromatography they are analyzed by tandem mass spectrometry and the substrates identified by database searching. The proof of principle in this study is demonstrated by incubating immobilized human plasma proteins with thrombin and by identifying by tandem mass spectrometry the fibrinopeptides, released by the action of thrombin from their natural substrate fibrinogen, in the reaction mixture

Fully automated forensic routine dried blood spot screening for workplace testing

3 Figuras, 3 Tablas.-- Sahar Y. Issa’s affiliation has been corrected to Faculty of Medicine, Alexandria University, Egypt.In this study, we describe the transfer of a new and fully automated workflow for the cost-effective drug screening of large populations based on the dried blood spot (DBS) technology. The method was installed at a routine poison control center and applied for DBS and dried urine spot (DUS) samples. A fast method focusing on the high-interest drugs and an extended screening method were developed on the automated platform. The dried cards were integrated into the automated workflow, in which the cards were checked in a camera recognition system, spiked with deuterated standards via an in-built spraying module and directly extracted. The extract was transferred online to an analytical LC column and then to the electrospray ionization tandem mass spectrometry system. The target compounds were analyzed in positive multiple-reaction monitoring mode. Before each sample batch or analysis day, calibration samples were measured to balance inter-day variations and to avoid false negative samples. An internal standard was integrated prior the sample extraction to allow in process control. A total of 28 target compounds were analyzed and directly extracted within 5 min per sample. This fast screening method was then extended to 20 min, enabling the usage of a Forensic Toxicology Database to screen over 1,200 drugs. The method gives confident positive/negative results for all tested drugs at their individual cut-off concentration. Good precision (±15%, respectively ±20% at limit of quantification) and correlation within the calibration range from 5 to 1,000 ng/mL was obtained. The method was finally applied to real cases from the lab and cross-checked with the existing methodologies.Peer reviewe

Fully automated forensic screening of dried blood spots with MRM Spectrum Mode

Work presented at the 67th ASMS Conference on Mass Spectrometry and Allied Topics, June 2nd-6th, 2019, Atlanta, GA (USA).-- MP 099.Fully automated forensic screening of dried bloodspots was achieved by coupling a DBS-MS 500 system (CAMAG, Switzerland) to an LCMS system (Shimadzu, Japan). An increasing amount of prescription and illicit drugs drive clinical laboratories to more cost effective and faster screening methods in workplace drug testing, roadside testing, rehabilitation programs or post-mortem investigations without compromising established false positive or negative detection rates. Therefore, the increasing sample amount with a large panel of analytes needs to be handled in shorter analysis time and with simple sample preparation steps. In this study we present a fully automated dried bloodspot (DBS) extraction procedure coupled to an LC-MS/MS-system for forensic and toxicology screening of drugs and their metabolites. We use a new approach, the MRM Spectrum Mode, by measuring at least 5 MRM-transitions of each compound to generate a high confidence for the identification of the results.Peer reviewe