Biomolecular Imaging Mass Spectrometry : mapping molecular distributions in cells and tissue sections

Abstract

Imaging mass spectrometry (IMS) allows the investigation of both identity and localization of the molecular content directly from tissue sections, single cells and many other surfaces. To further develop the application of IMS, different approaches to IMS will be described in this thesis and the specific sample preparation requirements will be discussed. Chapter 1 gives a general introduction of the biological systems under investigation and the specific biological questions asked. In chapter 2 the different ionization techniques (i.e. secondary ion mass spectrometry (SIMS) and matrix assisted laser desorption/ionization (MALDI)), the instrumentation and the different sample preparation techniques used in IMS are discussed. In chapter 3 we describe the necessary steps to retrieve the molecular information directly from tissue sections using ME-SIMS, MetA-SIMS and MALDI IMS in a protocol. Following these guidelines the different IMS methods offer a unique discovery tool in, for example, the investigation of (1) drug transport and uptake, (2) biological processing steps and (3) biomarker distribution. In order to extract the relevant information from the huge datasets produced by IMS, new bioinformatics solutions are described. Chapter 4 describes surface metallization by plasma coating, which enhances desorption/ionization of membrane components such as lipids and sterols in ToF-SIMS of tissues and cells. Alternatively, in matrix enhanced SIMS, 2,5-dihydroxybenzoic acid electrosprayed on neuroblastoma cells allowed intact molecular-ion imaging of phosphatidylcholine and sphingomyelin at the cellular level. Gold deposition on top of matrix-coated rat brain tissue sections strongly enhanced image quality and signal intensity in stigmatic matrix-assisted laser desorption/ionization imaging mass spectrometry. High-quality total ion count images were acquired and the neuropeptide vasopressin was localized in the rat brain tissue section at the hypothalamic area around the third ventricle. Chapter 5 describes in the first part the influence of the tissue microenvironment on the ionization efficiencies of different pseudomolecular ions of cholesterol. High-resolution images of cholesterol were obtained from Lymnaea stagnalis nervous tissue using Metal-Assisted (MetA) SIMS. The spatial distributions of these ions illustrate the influence of the biological matrix on the formation of specific pseudomolecular ions derived from the same molecular species. In the second part we used ME-SIMS for direct molecular imaging of nervous tissue at micrometer spatial resolution. High-resolution molecular ion maps of cholesterol and the neuropeptide APGWamide were constructed. APGWamide was predominantly localized in the cluster of neurons that regulate male copulation behavior of Lymnaea. ME-SIMS imaging allows direct molecule-specific imaging from tissue sections, without labelling and, opens a complementary mass window (<2500 Da) to MALDI imaging mass spectrometry, at an order of magnitude higher spatial resolution (<3 um). In Chapter 6 MALDI-IMS has been used to determine peptide distributions directly from rat, mouse and human pituitary tissue sections. Here we show that high-resolution IMS allows localization of neuropeptide distributions within different cell clusters of a single organ of a pituitary tissue section. Furthermore, we show that with imaging mass spectrometry a distinction can be made between different mammalian tissue sections based on differences in the amino acid sequence of neuropeptides with the same function.