Mass spectrometry based analysis of drugs, neurotransmitters and lipids in invertebrate model systems

Abstract

Mass spectrometry (MS) is one of the most universal analytical techniques due to its label-free detection principle and high chemical specificity, high selectivity, and sensitivity. MS is diverse with many different types of systems to meet different analytical demands from various research areas. MS can be used for bulk analysis, in particular when coupled with a separation tools such as capillary electrophoresis or liquid chromatography, provides highly accurate qualitative and quantitative information of sample compositions. Imaging mass spectrometry (IMS), on the other hand, allows for imaging the chemical structures in intact samples with impressive spatial resolution (< micron). In this thesis, MS is used for two main objectives. First, MS is used to investigate the concentration at the site of action of methylphenidate (MPH), and its neurological effects on the nervous system of Drosophila melanogaster (fruit fly). MPH, which is a common medical drug for attention deficit hyperactivity disorder and an alternative drug to replace cocaine during the process of quitting drug abuse, has a stimulant action similar to cocaine as it also binds to the dopamine transporter protein and thereby increases the concentration of extracellular dopamine, a neurotransmitter, in the mammalian nervous system. It has recently been evidence that MPH exhibits neurological impact in long-term use; however, the details of this disorder are not fully understood. Drosophila has been chosen as a model for these studies owing to its short life cycle, prolific reproduction, and highly conserved physiological effects with humans, especially in drug addiction. The second main objective of the thesis is to develop MSI methods for biomolecular imaging of tissue samples including Drosophila brain and C. elegans. Multimodal imaging with secondary ion mass spectrometry (SIMS) and laser desorption ionization mass spectrometry (LDI MS) of the fly brain provide complementary biomolecular information of the brain structure. The molecular signature of C. elegans, one of the primary biological models used today, is very useful for studies of cellular processes and can be related to behavior of the worm. In paper I, the in vivo concentration of MPH in Drosophila brain after oral administration was determined by capillary electrophoresis mass spectrometry (CE-MS). The information was then applied to study the effects of methylphenidate treatment on the action of cocaine on dopamine uptake in vivo in Drosophila. In paper II, capillary electrophoresis mass spectrometry was extensively used for qualitative and quantitative analysis of orally administrated methylphenidate and metabolites as well as evaluation of the drug-dose dependency of neurotransmitter concentrations in the fly brain. In paper III, an imaging protocol for Drosophila brain with SIMS, including sample preparation, data treatment with image-based principle components analysis, and continuous imaging was developed. The imaging protocol was applied in paper IV to investigate lipid structural effects of MPH on Drosophila brain. The distribution and biological functions of biomolecules in the fly brain are studied using a combination of SIMS and SEM imaging. In addition, it is demonstrated that oral administration of MPH significantly alters the distribution and abundance of various brain lipids. Paper V presents a multimodal imaging approach to Drosophila brain for lipid detection using matrix assisted laser desorption ionization (MALDI). Different surface modifications, including matrix sublimation and nanoparticle deposition, show specific detectable lipids and therefore can be used in a complementary fashion to profile biological samples. In paper VI, the chemical anatomy of C. elegans is studied using SIMS imaging. Two-dimensional and three-dimensional approaches are used for worm sections and the whole worm, respectively.