A Caged In-Source Laser-Cleavable MALDI Matrix with High Vacuum Stability for Extended MALDI-MS Imaging.

Insufficient vacuum stability of matrix chemicals is a major limitation in matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) of large tissue sample cohorts. Here, we designed and synthesized the photo-cleavable caged molecule 4,5-dimethoxy-2-nitrobenzyl-2,5-dihydroxyacetophenone (DMNB-2,5-DHAP) and employed it for lipid MALDI-MSI of mouse brain tissue sections. DMNB-2,5-DHAP is vacuum-stable in a high vacuum MALDI ion source for at least 72 h. Investigation of the uncaging process suggested that the built-in laser (355 nm) in the MALDI ion source promoted the in situ generation of 2,5-DHAP. A caging group is used for the first time in designing a MALDI matrix that is vacuum-stable, uncaged upon laser irradiation during the measurement process, and that boosts lipid ion intensity with MALDI-2 laser-induced postionization.

Recent developments of novel matrices and on-tissue chemical derivatization reagents for MALDI-MSI.

Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) is a fast-growing technique for visualization of the spatial distribution of the small molecular and macromolecular biomolecules in tissue sections. Challenges in MALDI-MSI, such as poor sensitivity for some classes of molecules or limited specificity, for instance resulting from the presence of isobaric molecules or limited resolving power of the instrument, have encouraged the MSI scientific community to improve MALDI-MSI sample preparation workflows with innovations in chemistry. Recent developments of novel small organic MALDI matrices play a part in the improvement of image quality and the expansion of the application areas of MALDI-MSI. This includes rationally designed/synthesized as well as commercially available small organic molecules whose superior matrix properties in comparison with common matrices have only recently been discovered. Furthermore, on-tissue chemical derivatization (OTCD) processes get more focused attention, because of their advantages for localization of poorly ionizable metabolites and their' in several cases' more specific imaging of metabolites in tissue sections. This review will provide an overview about the latest developments of novel small organic matrices and on-tissue chemical derivatization reagents for MALDI-MSI. Graphical abstract.

New Derivatization Reagent for Detection of free Thiol-groups in Metabolites and Proteins in Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging.

Several diseases are associated with disturbed redox signaling and altered metabolism of sulfur-containing metabolites and proteins. Importantly, oxidative degradation of fresh-frozen tissues begins within the normal time scale of MALDI MSI sample preparation. As a result, analytical methods that preserve the redox state of the tissue are urgently needed for refined studies of the underlying mechanisms. Nevertheless, no derivatization strategy for free sulfhydryl groups in tissue is known for MALDI MSI. Here, we report the first derivatization reagent, (E)-2-cyano-N-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)-3-(4-hydroxyphenyl)acrylamide (CHC-Mal), for selective detection of free thiols using MALDI MSI. We performed in situ derivatization of free thiol groups from thiol-containing metabolites such as glutathione and cysteine and reduced proteins such as insulin and imaged their spatial distribution in porcine and mouse xenograft tissue. Derivatization of thiol-containing metabolites with CHC-Mal for MALDI MSI was also possible when using aged tissue in the presence of excess reducing agents. Importantly, CHC-Mal-derivatized low mass-metabolites could be detected without the use of a conventional MALDI matrix.

Natural Products from Photorhabdus and Other Entomopathogenic Bacteria.

Although the first natural products (NP) from Photorhabdus and Xenorhabdus bacteria have been known now for almost 30 years, a huge variety of new compounds have been identified in the last 5-10 years, mainly due to the application of modern mass spectrometry. Additionally, application of molecular methods that allow the activation of NP production in several different strains as well as efficient heterologous expression methods have led to the production and validation of many new compounds. In this chapter we discuss the benefit of using Photorhabdus as a model system for microbial chemical ecology. We also examine non-ribosomal peptide synthetases as the most important pathway for NP production. Finally, we discuss the origin and function of all currently known NPs and the development of the molecular and chemical tools used to identify these NPs faster.

Simple "on-demand" production of bioactive natural products.

Exchange of the native promoter to the arabinose-inducible promoter PBAD was established in entomopathogenic bacteria to silence and/or activate gene clusters involved in natural product biosynthesis. This allowed the "on-demand" production of GameXPeptides, xenoamicins, and the blue pigment indigoidine. The gene clusters for the novel "mevalagmapeptides" and the highly toxic xenorhabdins were identified by this approach.

Biosynthesis of the insecticidal xenocyloins in Xenorhabdus bovienii.

The biosynthesis gene cluster for the production of xenocyloins was identified in the entomopathogenic bacterium Xenorhabdus bovienii SS-2004, and their biosynthesis was elucidated by heterologous expression and in vitro characterization of the enzymes. XclA is an S-selective ThDP-dependent acyloin-like condensation enzyme, and XclB and XclC are examples of the still-rare acylating ketosynthases that catalyze the acylation of the XclA-derived initial xenocyloins with acetyl-, propionyl-, or malonyl-CoA, thereby resulting in the formation of further xenocyloin derivatives. All xenocyloins were produced mainly by the more virulent primary variant of X. bovienii and showed activity against insect hemocytes thus contributing to the overall virulence of X. bovienii against insects.

Polyketide Trimming Shapes Dihydroxynaphthalene-Melanin and Anthraquinone Pigments.

Pigments such as anthraquinones (AQs) and melanins are antioxidants, protectants, or virulence factors. AQs from the entomopathogenic bacterium Photorhabdus laumondii are produced by a modular type II polyketide synthase system. A key enzyme involved in AQ biosynthesis is PlAntI, which catalyzes the hydrolysis of the bicyclic-intermediate-loaded acyl carrier protein, polyketide trimming, and assembly of the aromatic AQ scaffold. Here, multiple crystal structures of PlAntI in various conformations and with bound substrate surrogates or inhibitors are reported. Structure-based mutagenesis and activity assays provide experimental insights into the three sequential reaction steps to yield the natural product AQ-256. For comparison, a series of ligand-complex structures of two functionally related hydrolases involved in the biosynthesis of 1,8-dihydroxynaphthalene-melanin in pathogenic fungi is determined. These data provide fundamental insights into the mechanism of polyketide trimming that shapes pigments in pro- and eukaryotes.

Structure and biosynthesis of xenoamicins from entomopathogenic Xenorhabdus.

During the search for novel natural products from entomopathogenic Xenorhabdus doucetiae DSM17909 and X. mauleonii DSM17908 novel peptides named xenoamicins were identified in addition to the already known antibiotics xenocoumacin and xenorhabdin. Xenoamicins are acylated tridecadepsipeptides consisting of mainly hydrophobic amino acids. The main derivative xenoamicin A (1) was isolated from X. mauleonii DSM17908, and its structure elucidated by detailed 1D and 2D NMR experiments. Detailed MS experiments, also in combination with labeling experiments, confirmed the determined structure and allowed structure elucidation of additional derivatives. Moreover, the xenoamicin biosynthesis gene cluster was identified and analyzed in X. doucetiae DSM17909, and its participation in xenoamicin biosynthesis was confirmed by mutagenesis. Advanced Marfey's analysis of 1 showed that the absolute configuration of the amino acids is in agreement with the predicted stereochemistry deduced from the nonribosomal peptide synthetase XabABCD. Biological testing revealed activity of 1 against Plasmodium falciparum and other neglected tropical diseases but no antibacterial activity.

Xentrivalpeptides A-Q: depsipeptide diversification in Xenorhabdus.

Seventeen depsipeptides, xentrivalpeptides A-Q (1-17), have been identified from an entomopathogenic Xenorhabdus sp. Whereas the structure of xentrivalpeptide A (1) was determined after its isolation by NMR spectroscopy and the advanced Marfey's method, the structures of all other derivatives were determined using a combination of stable isotope labeling and detailed MS analysis.

Structural snapshots of the minimal PKS system responsible for octaketide biosynthesis.

Type II polyketide synthases (PKSs) are multi-enzyme complexes that produce secondary metabolites of medical relevance. Chemical backbones of such polyketides are produced by minimal PKS systems that consist of a malonyl transacylase, an acyl carrier protein and an α/ß heterodimeric ketosynthase. Here, we present X-ray structures of all ternary complexes that constitute the minimal PKS system for anthraquinone biosynthesis in Photorhabdus luminescens. In addition, we characterize this invariable core using molecular simulations, mutagenesis experiments and functional assays. We show that malonylation of the acyl carrier protein is accompanied by major structural rearrangements in the transacylase. Principles of an ongoing chain elongation are derived from the ternary complex with a hexaketide covalently linking the heterodimeric ketosynthase with the acyl carrier protein. Our results for the minimal PKS system provide mechanistic understanding of PKSs and a fundamental basis for engineering PKS pathways for future applications.

Molecular mechanism of polyketide shortening in anthraquinone biosynthesis of Photorhabdus luminescens.

Anthraquinones, a widely distributed class of aromatic natural products, are produced by a type II polyketide synthase system in the Gram-negative bacterium Photorhabdus luminescens. Heterologous expression of the antABCDEFGHI anthraquinone biosynthetic gene cluster in Escherichia coli identified AntI as an unusual lyase, catalysing terminal polyketide shortening prior to formation of the third aromatic ring. Functional in vitro and in vivo analysis of AntI using X-ray crystallography, structure-based mutagenesis, and molecular simulations revealed that AntI converts a defined octaketide to the tricyclic anthraquinone ring via retro-Claisen and Dieckmann reactions. Thus, AntI catalyses a so far unobserved multistep reaction in this PKS system.