Ambient laser ablation electrospray ionization mass spectrometry imaging

Several molecular imaging techniques are available to study and understand biological objects, like positron-emission tomography, fluorescence, and magnetic resonance imaging. These techniques often require chemical probes and image in a targeted approach. As many biological questions can only be answered in a systems approach, molecular imaging methods that can simultaneously measure many molecules are desired. Mass spectrometry imaging (MSI) is capable of measuring many molecules simultaneously without the use of chemical probes. MSI experiments often require sample stage vacuum conditions and extensive sample pretreatment such as matrix application. Vacuum conditions can disrupt or damage biological samples, and sample pretreatment prevents real-time analyses and can cause analyte losses, analyte delocalization and denaturation of proteins. Ambient ionization was introduced to measure samples under ambient conditions without any sample pretreatment, and ambient MSI followed rapidly after that. A next generation of ambient MSI techniques is desired to improve its sensitivity, molecular mass range, and spatial resolution. This work aims to improve the capabilities and broaden the scope of laser ablation electrospray ionization (LAESI) MSI. In order to broaden the scope and increase the understanding of ambient LAESI-MS(I), polymer materials and synthetic fibers were investigated. The direct analysis of synthetic fibers under ambient conditions is highly desired to identify the polymer, the finishes applied and irregularities that may compromise its performance and value. In Chapter 2 LAESI ion mobility MS was used for the analysis of synthetic polymers and fibers. The key to this analysis was the absorption of laser light by aliphatic and aromatic nitrogen functionalities in the polymers. Analysis of polyamide (PA) 6, 46, 66, and 12 pellets and PA 6, 66, polyaramid and M5 fibers yielded characteristic fragment ions, enabling their unambiguous identification. Synthetic fibers are, in addition, commonly covered with a surface layer for improved adhesion and processing. The same setup, but operated in a transient infrared matrix-assisted laser desorption electrospray ionization mode, allowed the detailed characterization of the fiber finish layer and the underlying polymer. Differences in finish layer distribution may cause variations in local properties of synthetic fibers. In Chapter 2, also the feasibility of mass spectrometry imaging (MSI) of the distribution of a finish layer on the synthetic fiber and the successful detection of local surface defects was shown. Reactions in confined compartments like charged microdroplets are of increasing interest, notably because of their substantially increased reaction rates. When combined with ambient MS, reactions in charged microdroplets can be used to improve the detection of analytes or to study the molecular details of the reactions in real time. In Chapter 3, we introduce a reactive LAESI time-resolved MS method to perform and study reactions in charged microdroplets. This approach was demonstrated with so-called click chemistry reactions between substituted tetrazines and a strained alkyne or alkene. Click reactions are high-yielding reactions with a high atom efficiency. Although click reactions are typically at least moderately fast, in a reactive LAESI approach a substantial increase of reaction time is Summary 149 required for these reactions to occur. This increase was achieved using microdroplet chemistry and followed by MS using the insertion of a reaction tube between the LAESI source and the MS inlet, leading to near complete conversions due to significantly extended microdroplet lifetime. This novel approach allowed for the collection of kinetic data for a model click reaction and showed in addition excellent instrument stability, improved sensitivity, and applicability to other click reactions. In Chapter 3, reactive LAESI was also demonstrated in a mass spectrometry imaging setting to show its feasibility in future imaging experiments. In drug discovery it is important to identify phase I metabolic modifications as early as possible to screen for inactivation of drugs and/or activation of prodrugs. As the major class of reactions in phase I metabolism are oxidation reactions, oxidation of drugs with TiO2 photocatalysis can be used as a simple non-biological method to initially eliminate (pro)drug candidates with an undesired phase I oxidation metabolism. Analysis of reaction products is commonly achieved with mass spectrometry coupled to chromatography. However, sample throughput can be substantially increased by eliminating pretreatment steps and exploiting the potential of ambient MS. Furthermore, online monitoring of reactions in a time-resolved way would identify sequential modification steps. In Chapter 4 we introduce a novel (time-resolved) TiO2-photocatalysis LAESI-MS method for the analysis of drug candidates. This method was proven to be compatible with both TiO2-coated glass slides as well as solutions containing suspended TiO2 nanoparticles, and the results were in excellent agreement with studies on biological oxidation of several drugs. Additionally, a time-resolved LAESI-MS setup was developed and results for verapamil showed excellent analytical stability for online photocatalyzed oxidation reactions within the set-up up to at least one hour. Identification and confirmation of (bio)chemical entities in ambient MS mostly involves accurate mass determination, often in combination with MS/MS work flows. However, an accurate mass only provides the elemental composition of the (bio)molecule, still resulting in numerous possible structures. MS/MS procedures are often insufficient in differentiating between the hundreds possible candidate substances in database searches. Obtaining additional information and thereby improving structural assignment as well as reducing the vast number of possible candidates is thus of high importance in any ambient MS(I) study. In Chapter 5 we present an ambient hydrogen/deuterium exchange (HDX) LAESI-MS method for structure elucidation and confirmation of (bio)molecules. The concept was demonstrated with small molecules, peptides, and proteins. Moreover, the same approach could be applied to MSI as shown by the ambient MSI of arginine and oligosaccharides on an orange slice. Eventually, this approach will allow spatially resolved MSI of different protein conformers and may have a major impact in the life sciences. The main achievements that are described in this thesis offer insights on sample compatibility, hardware improvements to enable online time-resolved reactions and structure elucidation approaches. The outcome of the research chapters shows that LAESI-MS(I) is a highly versatile technique applicable to many research areas. Although the technique is highly 150 dependent on endogenous water in samples for analysis of intact molecules, LAESI can also be exploited for the analysis and identification of (water free) polymer materials. Unfortunately, LAESI sensitivity relative to electrospray ionization is weak, and therefore the technique can currently not live up to the status of the next generation of ambient MSI. Analytes that are present in high abundance are feasible for imaging by LAESI-MS. For low abundance analytes, however, several hardware improvements are required to substantially increase the sensitivity of the results. When the hardware improvements are developed and implemented, the road is open for many end users in, e.g., microbiology, pathology, and botany, to make significant breakthroughs in their fields.