Multigrid MALDI mass spectrometry imaging (mMALDI MSI).

Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) is an important technique for the spatially resolved molecular analysis of tissue sections. The selection of matrices influences the resulting mass spectra to a high degree. For extensive and simultaneous analysis, the application of different matrices to one tissue sample is desirable. To date, only a single matrix could be applied to a tissue section per experiment. However, repetitive removal of the matrix makes this approach time-consuming and damaging to tissue samples. To overcome these drawbacks, we developed a multigrid MALDI MSI technique (mMALDI MSI) that relies on automated inkjet printing to place differing matrices onto predefined dot grids. We used a cooled printhead to prevent cavitation of low viscosity solvents in the printhead nozzle. Improved spatial resolution of the dot grids was achieved by using a triple-pulse procedure that reduced droplet volume. The matrices can either be applied directly to the thaw-mounted tissue sample or by precoating the slide followed by mounting of the tissue sample. During the MALDI imaging process, we were able to precisely target different matrix point grids with the laser to simultaneously produce distinct mass spectra. Unlike the standard method, the prespotting approach optimizes the spectra quality, avoids analyte delocalization, and enables subsequent hematoxylin and eosin (H&E) staining. Graphical Abstract Scheme of the pre-spotted multigrid MALDI MSI workflow.

High resolution mass spectrometry imaging reveals the occurrence of phenylphenalenone-type compounds in red paracytic stomata and red epidermis tissue of Musa acuminata ssp. zebrina cv. 'Rowe Red'.

The banana epidermis and in particular their stomata are conducive sites for the penetration of pathogenic fungi which can severely limit global banana production. The red pseudostem of the ornamental banana Musa acuminata ssp. zebrina cv. 'Rowe Red' was used to study the chemical constituents of the epidermal cell layer using matrix-free laser desorption/ionization Fourier transform ion cyclotron resonance mass spectrometric imaging (LDI-FT-ICR-MSI). The high resolution of this technique allowed phenylphenalenone-type compounds to be located in single plant cells. Some of these secondary metabolites were identified as constitutive compounds and found in specialized epidermal cells in banana pseudostem tissue. Especially the red paracytic stomata revealed higher signal intensities of certain phenylphenalenones than normal epidermis cells. The ease of detection of polycyclic aromatic compounds on the cellular level is discussed with regard to future investigations of plant-pathogen interactions.

COCONUT­An Efficient Tool for Estimating Copolymer Compositions from Mass Spectra.

The accurate characterization of synthetic polymer sequences represents a major challenge in polymer science. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is frequently used for the characterization of copolymer samples. We present the COCONUT software for estimating the composition distribution of the copolymer. Our method is based on Linear Programming and is capable of automatically resolving overlapping isotopes and isobaric ions. We demonstrate that COCONUT is well suited for analyzing complex copolymer MS spectra. COCONUT is freely available and provides a graphical user interface.

Phenalenone-type phytoalexins mediate resistance of banana plants (Musa spp.) to the burrowing nematode Radopholus similis.

The global yield of bananas-one of the most important food crops-is severely hampered by parasites, such as nematodes, which cause yield losses up to 75%. Plant-nematode interactions of two banana cultivars differing in susceptibility to Radopholus similis were investigated by combining the conventional and spatially resolved analytical techniques (1)H NMR spectroscopy, matrix-free UV-laser desorption/ionization mass spectrometric imaging, and Raman microspectroscopy. This innovative combination of analytical techniques was applied to isolate, identify, and locate the banana-specific type of phytoalexins, phenylphenalenones, in the R. similis-caused lesions of the plants. The striking antinematode activity of the phenylphenalenone anigorufone, its ingestion by the nematode, and its subsequent localization in lipid droplets within the nematode is reported. The importance of varying local concentrations of these specialized metabolites in infected plant tissues, their involvement in the plant's defense system, and derived strategies for improving banana resistance are highlighted.

Drugs as matrix to detect their own drug delivery system of PEG-b-PCL block copolymers in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

RATIONALE: The fate of drug delivery systems (DDSs) in vivo is a widely discussed question. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is an important tool to localize proteins and small compounds in many different tissues. This basic study was performed as an aid to obtain spatial information on DDSs in the future. METHODS: LDI and MALDI-TOF MS was used to investigate five drug molecules, i.e. madurahydroxylactone (MHL), tetrakis(4-hydroxyphenyl)porphyrin (THP), chartreusin (Chart), amphotericin B (AmB) and retinoic acid (RA). The drug molecules were analyzed in terms of their efficiency to act as matrix for different homopolymers and the block copolymer poly(ethylene glycol)-block-poly(ε-caprolactone) (PEG-b-PCL). The block copolymer was further utilized as a DDS to encapsulate the drug molecules previously investigated as matrices. The obtained DDSs were investigated by MALDI. RESULTS: The spectra obtained with the drugs Chart, MHL, THP and AmB did not reach the quality of the standard matrix RA. Nonetheless, they showed surprisingly good results as matrices for different homopolymers and the block copolymer. However, only the DDSs containing THP as the drug provided spectra where the drug and the block copolymer were detected. CONCLUSIONS: These results form the foundation for obtaining mass-related information about the localization of DDSs and drugs in tissues.

Star-shaped drug carriers for doxorubicin with POEGMA and POEtOxMA brush-like shells: a structural, physical, and biological comparison.

The synthesis of amphiphilic star-shaped poly(ε-caprolactone)-block-poly(oligo(ethylene glycol)methacrylate)s ([PCL(18)-b-POEGMA](4)) and poly(ε-caprolactone)-block-poly(oligo(2-ethyl-2-oxazoline)methacrylate)s ([PCL(18)-b-POEtOxMA](4)) is presented. Unimolecular behavior in aqueous systems is observed with the tendency to form loose aggregates for both hydrophilic shell types. The comparison of OEGMA and OEtOxMA reveals that the molar mass of the macromonomer in the hydrophilic shell rather than the mere length is the crucial factor to form an efficiently stabilizing hydrophilic shell. A hydrophilic/lipophilic balance of 0.8 is shown to stabilize unimolecular micelles in water. An extensive in vitro biological evaluation shows neither blood nor cytotoxicity. The applicability of the polymers as drug delivery systems was proven by the encapsulation of the anticancer drug doxorubicin, whose cytotoxic effect was retarded in comparison to the free drug.

4-Deoxyaurone formation in Bidens ferulifolia (Jacq.) DC.

The formation of 4-deoxyaurones, which serve as UV nectar guides in Bidens ferulifolia (Jacq.) DC., was established by combination of UV photography, mass spectrometry, and biochemical assays and the key step in aurone formation was studied. The yellow flowering ornamental plant accumulates deoxy type anthochlor pigments (6'-deoxychalcones and the corresponding 4-deoxyaurones) in the basal part of the flower surface whilst the apex contains only yellow carotenoids. For UV sensitive pollinating insects, this appears as a bicoloured floral pattern which can be visualized in situ by specific ammonia staining of the anthochlor pigments. The petal back side, in contrast, shows a faintly UV absorbing centre and UV absorbing rays along the otherwise UV reflecting petal apex. Matrix-free UV laser desorption/ionisation mass spectrometric imaging (LDI-MSI) indicated the presence of 9 anthochlors in the UV absorbing areas. The prevalent pigments were derivatives of okanin and maritimetin. Enzyme preparations from flowers, leaves, stems and roots of B. ferulifolia and from plants, which do not accumulate aurones e.g. Arabidopsis thaliana, were able to convert chalcones to aurones. Thus, aurone formation could be catalyzed by a widespread enzyme and seems to depend mainly on a specific biochemical background, which favours the formation of aurones at the expense of flavonoids. In contrast to 4-hydroxyaurone formation, hydroxylation and oxidative cyclization to the 4-deoxyaurones does not occur in one single step but is catalyzed by two separate enzymes, chalcone 3-hydroxylase and aurone synthase (catechol oxidase reaction). Aurone formation shows an optimum at pH 7.5 or above, which is another striking contrast to 4-hydroxyaurone formation in Antirrhinum majus L. This is the first example of a plant catechol oxidase type enzyme being involved in the flavonoid pathway and in an anabolic reaction in general.

Tandem mass spectrometry of poly(ethylene imine)s by electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI).

In this contribution, linear poly(ethylene imine) (PEI) polymers, which are of importance in gene delivery, are investigated in detail by using electrospray ionization-quadrupole-time of flight (ESI-Q-TOF) and matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry (MS). The analyzed PEIs with different end groups were synthesized using the polymerization of substituted 2-oxazoline via a living cationic ring-opening polymerization (CROP) and a subsequent hydrolysis under acidic conditions. The main goal of this study was to identify linear PEI polymers in a detailed way to gain information about their fragmentation pathways. For this purpose, a detailed characterization of three different linear PEIs was performed by using ESI-Q-TOF and MALDI-TOF MS in combination with collision-induced dissociation (CID) experiments. In ESI-MS as well as MALDI-MS analysis, the obtained spectra of PEIs resulted in fitting mass distributions for the investigated PEIs. In the tandem MS analysis, a 1,2-hydride shift with a charge-remote rearrangement via a four-membered cyclic transition state, as well as charge-induced fragmentation reactions, was proposed as the main fragmentation mechanisms according to the obtained fragmentation products from the protonated parent peaks. In addition, heterolytic and homolytic cleavages were proposed as alternative fragmentation pathways. Moreover, a 1,4-hydrogen elimination was proposed to explain different fragmentation products obtained from the sodiated parent peaks.

Poly(ethylene glycol) in drug delivery: pros and cons as well as potential alternatives.

Poly(ethylene glycol) (PEG) is the most used polymer and also the gold standard for stealth polymers in the emerging field of polymer-based drug delivery. The properties that account for the overwhelming use of PEG in biomedical applications are outlined in this Review. The first approved PEGylated products have already been on the market for 20 years. A vast amount of clinical experience has since been gained with this polymer--not only benefits, but possible side effects and complications have also been found. The areas that might need consideration and more intensive and careful examination can be divided into the following categories: hypersensitivity, unexpected changes in pharmacokinetic behavior, toxic side products, and an antagonism arising from the easy degradation of the polymer under mechanical stress as a result of its ether structure and its non-biodegradability, as well as the resulting possible accumulation in the body. These possible side effects will be discussed in this Review and alternative polymers will be evaluated.

Matrix-free UV-laser desorption/ionization (LDI) mass spectrometric imaging at the single-cell level: distribution of secondary metabolites of Arabidopsis thaliana and Hypericum species.

The present paper describes matrix-free laser desorption/ionisation mass spectrometric imaging (LDI-MSI) of highly localized UV-absorbing secondary metabolites in plant tissues at single-cell resolution. The scope and limitations of the method are discussed with regard to plants of the genus Hypericum. Naphthodianthrones such as hypericin and pseudohypericin are traceable in dark glands on Hypericum leaves, placenta, stamens and styli; biflavonoids are also traceable in the pollen of this important phytomedical plant. The highest spatial resolution achieved, 10 microm, was much higher than that achieved by commonly used matrix-assisted laser desorption/ionization (MALDI) imaging protocols. The data from imaging experiments were supported by independent LDI-TOF/MS analysis of cryo-sectioned, laser-microdissected and freshly cut plant material. The results confirmed the suitability of combining laser microdissection (LMD) and LDI-TOF/MS or LDI-MSI to analyse localized plant secondary metabolites. Furthermore, Arabidopsis thaliana was analysed to demonstrate the feasibility of LDI-MSI for other commonly occurring compounds such as flavonoids. The organ-specific distribution of kaempferol, quercetin and isorhamnetin, and their glycosides, was imaged at the cellular level.

Monomeric pheophorbide(a)-containing poly(ethyleneglycol-b-epsilon-caprolactone) micelles for photodynamic therapy.

Incorporation of unaggregated monomeric molecules of pheophorbide(a) into micelles of poly(ethyleneoxide-b-epsilon-caprolactone) has been characterized by fluorescence spectroscopy, dynamic light scattering, transmission electron microscopy and asymmetric flow field flow fractionation. It was shown that the method used leads to 20 nm micelles, corresponding to approximately 200 molecules of polymer and 4 molecules of monomeric pheophorbide(a) per nano-object which was able to generate (1)O(2) in the medium. They have been used thereafter as nanocarriers for photosensitizers in photodynamic therapy against cancer cells. The encapsulation of photosensitizer has been verified and in vitro tests on human cancerous cells have revealed a ca. 2-fold enhanced photocytotoxicity and cellular uptake compared to free pheophorbide(a).

Characterization of different poly(2-ethyl-2-oxazoline)s via matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) coupled with CID (collision-induced dissociation) has been used for the detailed characterization of two poly(2-ethyl-2-oxazoline)s as part of a continuing study of synthetic polymers by MALDI-TOF MS/MS. These experiments provided information about the variety of fragmentation pathways for poly(oxazoline)s. It was possible to show that, in addition to the eliminations of small molecules, like ethene and hydrogen, the McLafferty rearrangement is also a possible fragmentation route. A library of fragmentation pathways for synthetic polymers was also constructed and such a library should enable the fast and automated data analysis of polymers in the future.

Mesoionic bora-tetraazapentalenes--fully reversible two step redox systems.

Bora-tetraazapentalenes have been synthesised and characterized; the radical anion shows an unusual thermodynamic stability (semiquinone formation constants K(SEM) of ca. 10(14)).