Self-Classified Topic Area(s): Small Molecules / Tox / TDM
LC-MS/MS for Peptide-Adduct Analysis to Verify Poisoning with Chemical Warfare Agents and Pesticides
Harald John Bundeswehr Institute of Pharmacology & Toxicology
Warning: Undefined variable $headshot in /var/www/html/view_abstract/view_abstract_in_program.php on line 704
Harald John (Presenter) Bundeswehr Institute of Pharmacology & Toxicology
Presenter Bio: Harald John studied chemistry at the Westfälische-Wilhelms University of Münster (Germany). In 1998 he finished his Ph.D. studies in the group of K. Cammann and W. Schlegel in Analytical Chemistry on the development and validation of immunological and chromatographic procedures for the determination of prostaglandins and related compounds in clinical samples. Following a period as a research associate at the Institute of Occupational Medicine, University of Münster, he moved to the Lower Saxony Institute for Peptide Research, Hannover (Germany) for a postdoctoral fellowship. From 2000 to 2005 Harald John has been Head of the Division of Analytical Peptide Chemistry at IPF PharmaCeuticals GmbH, Hannover. The analytical division was responsible for the discovery, detection and structural identification of bioactive peptides from natural sources using combinations of immunological, chromatographic, capillary electrophoretic, and in particular mass spectrometric techniques. Based on this progress Harald John has habilitated (postdoctoral university lecturing qualification) at the Humboldt-Universität zu Berlin (Germany) in 2007 as an external scientist and pursues his venia legendi (assistant professor) in Analytical Chemistry at the same university. Since 2016 he is professor at that university. Later on, he additionally studied toxicology at the Faculty of Medicine at the University of Leipzig (Germany) and finished his degree as a scientific specialist for toxicology in 2012. In the beginning of 2006 he changed to the Bundeswehr Institute of Pharmacology and Toxicology, Munich (Germany) - a national institution instructed to develop novel medical countermeasures and to improve therapeutic regimen for the treatment of poisoning with chemical warfare agents (CWA) and related toxicants. Harald John is heading a working group now focused on the development of analytical procedures applied to biological samples with toxicological, forensic or clinical relevance to determine highly toxic CWA and related organophosphorus pesticides as well as their relevant therapeutics and antidotes.
Relevant Financial Disclosures
(within past 24 months)
No relevant financial relationship(s) to disclose.
Abstract
Introduction:
Chemical warfare agents (CWA) have been used in recent years in a variety of military conflicts as well as in terroristic attacks at the airport in Kuala Lumpur, Malaysia and in Salisbury, UK. Current relevance is attributed to nerve agents like sarin or the skin-active vesicants like sulfur mustard (SM). Since the use of CWAs is banned by the chemical weapons convention (CWC) it is strictly required for criminal prosecution to verify poisoning by CWAs through unambigous biomarker detection and verification.
Objectives:
This study describes the development and the application of a set of analytical methods to detect CWA-peptide-adducts as biomarkers of CWA poisoning by LC-MS/MS. Such adducts are formed as covalent reaction products of the CWA with endogeneous proteins like human serum albumin (HSA) or butyrylcholinesterase (BChE).
Methods:
Specific peptide-adduct biomarkers were developed from their corresponding proteins by plasma proteolysis using various proteases. Subsequently, separation and detection were carried out by selective micro-liquid chromatography ionization high-resolution tandem-mass spectrometry (LC-ESI MS/HR MS).
Results:
SM and nerve-agent-mediated poisoning in human samples was determined by detection of diverse albumin-derived and butyrylcholinesterase-derived protein adducts.
In case of SM, the SM-specific hydroxyethylthioethyl moiety bound to the thiol group of HSA Cys34 (C34*) or to the side-chain carboxylic group of HSA Glu230 (E230*) was used for detection. The corresponding peptide biomarkers were generated by proteinase K (C34*PF), pronase (C34*P), and pepsin (AE230*VSKL and LQQC34*PFEDHVKL) cleavage.
The presence of organophosphorus nerve agents and pesticides was also proven by the detection of their adducts with HAS as well as with BChE.
Discussion:
The methods developed provided unambiguous forensic evidence of CWA- and pesticide-exposure in real cases of human poisoning. Due to their long half-live of some weeks up to some months targeted protein-adducts are highly important post-exposure biomarkers beneficial for verification purposes.
= Discovery stage.
= Translation stage.
= Clinically available.
