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Microglia are non-neuronal cells, which are residing in the central nervous system and are known to play an important role in health and disease. We investigated lipidomic phenotypes of human naïve and stimulated microglia-like cells by atmospheric-pressure scanning microprobe matrix-assisted laser desorption/ionization mass spectrometry imaging (AP-SMALDI MSI). With lateral resolutions between 5 μm and 1.5 μm, we were able to chart lipid compositions of individual cells, enabling to differentiate cell lines and stimulation conditions. This allowed us to reveal local lipid heterogeneities in naïve and lipopolysaccharide (LPS)-stimulated cells. We were able to identify individual cells with elevated triglyceride (TG) levels and could show that the number of these TG-enriched cells increased with LPS stimulation as a hallmark for a proinflammatory phenotype. Additionally, observed local abundance alterations of specific phosphatidylinositols (PIs) indicate a cell specific regulation of the PI metabolism.

The snake venom gland is the place for the synthesis, storage, and secretion of a complex mixture of proteins and peptides, i.e., the venom. The morphology of the gland has been revealed by classical histology and microscopic studies. However, knowledge about the gland's cellular secretory and functional processes is still incomplete and has so far been neglected by the omics disciplines. We used autofocusing atmospheric-pressure matrix-assisted laser desorption/ionization (AP-SMALDI) mass spectrometry imaging (MSI) to investigate endogenous biomolecular distributions in the venom glands of the saw-scaled viper, Echis carinatus sochureki, employing different sample preparation methods. Fresh-freezing and formalin-fixation were tested for the gland to obtain intact tissue sections. Subsequently, MSI was conducted with 12 μm pixel resolution for both types of preparations, and the lateral distributions of the metabolites were identified. Experiments revealed that lipids belonging to the classes of PC, SM, PE, PS, PA, and TG are present in the venom gland. PC (32:0) and SM (36:1) were found to be specifically located in the areas where cells are present. The snake venom metalloprotease inhibitor pEKW (m/z 444.2233) was identified in the venom by top-down LC-MS/MS and localized by MALDI-MSI in the gland across secretory epithelial cells. The peptide can inhibit the venom's enzymatic activity during long-term storage within the venom gland. With a high degree of spectral similarities, we concluded that formalin-fixed tissue, in addition to its high ability to preserve tissue morphology, can be considered as an alternative method to fresh-frozen tissue in the case of lipid and peptide MS imaging in venom gland tissues.

Abstract Aldehydes represent a versatile and favored class of flavoring substances. A biocatalytic access to odor-active aldehydes was developed by conversion of fatty acids with two enzymes of the α-dioxygenase pathway. The recombinant enzymes α-dioxygenase (α-DOX) originating from Crocosphaera subtropica and fatty aldehyde dehydrogenase (FALDH) from Vibrio harveyi were heterologously expressed in E. coli, purified, and applied in a coupled (tandem) repetitive reaction. The concept was optimized in terms of number of reaction cycles and production yields. Up to five cycles and aldehyde yields of up to 26% were achieved. Afterward, the approach was applied to sea buckthorn pulp oil as raw material for the enzyme catalyzed production of flavoring/fragrance ingredients based on complex aldehyde mixtures. The most abundant fatty acids in sea buckthorn pulp oil, namely palmitic, palmitoleic, oleic, and linoleic acid, were used as substrates for further biotransformation experiments. Various aldehydes were identified, semi-quantified, and sensorially characterized by means of headspace–solid phase microextraction–gas chromatography–mass spectrometry–olfactometry (HS–SPME–GC–MS–O). Structural validation of unsaturated aldehydes in terms of double-bond positions was performed by multidimensional high-resolution mass spectrometry experiments of their Paternò–Büchi (PB) photoproducts. Retention indices and odor impressions of inter alia (Z,Z)-5,8-tetradecadienal (Z,Z)-6,9-pentadecadienal, (Z)-8-pentadecenal, (Z)-4-tridecenal, (Z)-6-pentadecenal, and (Z)-8-heptadecenal were determined for the first time. Key points • Coupled reaction of Csα-DOX and VhFALDH yields chain-shortened fatty aldehydes. • Odors of several Z-unsaturated fatty aldehydes are described for the first time. • Potential for industrial production of aldehyde-based odorants from natural sources. Graphical abstract

Matrix-assisted laser desorption/ionization mass spectrometry imaging is a promising tool in the life sciences for obtaining spatial and chemical information from complex biological samples. State-of-the-art setups combine high mass resolution and high mass accuracy with high lateral resolution, offering untargeted insights into biochemical processes on the single-cell length scale. Despite recent technological breakthroughs, the sensitivity and acquisition speed of many setups are often in competition with achievable pixel resolutions below 25 μm. New measurement modes were developed by implementing a high-repetition-rate laser into an AP-SMALDI10 ion source, coupled to an orbital trapping mass spectrometer. These new MSI modes allow for a modular use of the new setup. We demonstrate that the system allows single cell features to be visualized in mouse brain tissue sections at a pixel resolution of 5 μm and an imaging speed of 18 pixels/s. Furthermore, the analytical sensitivity was improved in another measurement mode by applying multiple pulses of a highly focused laser beam over larger square pixels ≥25 μm edge length, increasing ion signal intensities up to 20-fold on tissue and decreasing the limit of detection by 1 order of magnitude.

Local lipid variations in tissues are readily revealed with mass spectrometry imaging (MSI) methods, and the resulting lipid distributions serve as bioanalytical signatures to reveal cell- or tissue-specific lipids. Comprehensive MSI lipid mapping requires measurements in both ion polarities. Additionally, structural lipid characterization is necessary to link the lipid structure to lipid function. Whereas some structural elements of lipids are readily derived from high-resolution mass spectrometry (MS) and tandem-MS (MSn), the localization of C═C double bonds (DBs) requires specialized fragmentation and/or functionalization methods. In this work, we identify a multifunctional matrix-assisted laser desorption/ionization (MALDI) matrix for spatially resolved lipidomics investigations that reacts with lipids in Paterno-Buchi (PB) reactions during laser irradiation facilitating DB-position assignment and allows dual-polarity high-resolution MALDI-MSI and MALDI MS2I studies. By screening 12 compounds for improved ionization efficiency in positive-/negative-ion mode and the functionalization yield compared to the previously introduced reactive MALDI matrix benzophenone, 2-benzoylpyridine (BzPy) is identified as the best candidate. The new matrix enables DB localization of authentic standards belonging to 12 lipid classes and helps to assign 133/58 lipid features in positive-/negative-ion mode from mouse cerebellum tissue. The analytical capabilities of BzPy as a multifunctional MALDI-MSI matrix are demonstrated by imaging endogenous and PB-functionalized lipids in mouse kidney sections with 7 μm lateral resolution in both ion modes. Tracking diagnostic lipid DB-position fragment ions in mouse pancreatic tissue with down to 10 μm pixel size allows us to identify the islets of Langerhans associated with lipid isomer upregulation and depletion.

CeO2 is a promising material in the catalytic recovery of Cl2 from HCl by oxidation with O2 due to its beneficial redox properties (Ce3+/Ce4+) and accompanied oxygen storage capacity (OSC). Two options to tailor the OSC are the usage of dopants and nanostructuring. In this work, we report the synthesis of mesoporous doped Ce0.9M0.1O2 (M = Zr, Gd, Pr, and Tb) powders with identical mesopore size, shape, and specific surface area (≈90 m2 g– 1) to investigate the impact of aliovalent (Gd), isovalent (Zr), and aliovalent/isovalent (Pr and Tb) dopants on the OSC as well as on the catalytic activity/stability. The cubic arrangement of spherical 12 nm mesopores surrounded by a crystalline framework is obtained by using a diblock copolymer as structure directing agent in the sol-gel-based preparation approach, indicated by small angle X-ray scattering (SAXS) and scanning electron microscopy (SEM). Pr-doped CeO2 revealed the highest oxygen storage capacity but the lowest catalytic activity/stability in the HCl oxidation together with the Tb- and Gd-doped samples. By contrast, the Zr-doped mesoporous CeO2 powders showed the highest catalytic performance and stability in the CO and HCl oxidation of all samples but a lower OSC. These findings suggest that the OSC should therefore be treated with caution when relating to the catalytic performance in oxidation reactions. The differences in the stability are in agreement with recent studies of the HCl oxidation on model cerium oxide films with defined stoichiometry: the low stability of the aliovalent doped CeO2 materials in the HCl oxidation reaction can be attributed to a large number of oxygen vacancies.

Spatial metabolomics describes the spatially resolved analysis of interconnected pathways, biochemical reactions, and transport processes of small molecules in the spatial context of tissues and cells. However, a broad range of metabolite classes (e.g., steroids) show low intrinsic ionization efficiencies in mass spectrometry imaging (MSI) experiments, thus restricting the spatial characterization of metabolic networks. Additionally, decomposing complex metabolite networks into chemical compound classes and molecular annotations remains a major bottleneck due to the absence of repository-scaled databases. Here, we describe a multimodal mass-spectrometry-based method combining computational metabolome mining tools and high-resolution on-tissue chemical derivatization (OTCD) MSI for the spatially resolved analysis of metabolic networks at the low micrometer scale. Applied to plant toxin sequestration in

Ambient mass spectrometry imaging (MSI) methods come with the advantage of visualizing biomolecules from tissues with no or minimal sample preparation and operation under atmospheric-pressure conditions. Similar to all other MSI methodologies, however, ambient MSI modalities suffer from a pronounced bias toward either polar or nonpolar analytes due to the underlying desorption and ionization mechanisms of the ion source. In this study, we present the design, construction, testing, and application of an in-capillary dielectric barrier discharge (DBD) module for post-ionization of neutrals desorbed by an ambient infrared matrix-assisted laser desorption/ionization (IR-MALDI) MSI source. We demonstrate that the DBD device enhances signal intensities of nonpolar compounds by up to 104 compared to IR-MALDI without affecting transmission of IR-MALDI ions. This allows performing MSI experiments of mouse tissue and Danaus plexippus caterpillar tissue sections, visualizing the distribution of sterols, fatty acids, monoglycerides, and diglycerides that are not detected in IR-MALDI MSI experiments. The pronounced signal enhancement due to IR-MALDI-DBD compared to IR-MALDI MSI enables mapping of nonpolar analytes with pixel resolutions down to 20 μm in mouse brain tissue and to discern the spatial distribution of sterol lipids characteristic for histological regions of D. plexippus.

Glycerophospholipids (GPs) are highly abundant in eukaryotic cells and take part in numerous fundamental physiological processes such as molecular signaling. The GP composition of samples is often analyzed using mass spectrometry (MS), but identification of some structural features, for example, differentiation of stereospecific numbering (

This contribution is part of our ongoing efforts to develop innovative cross-linking (XL) reagents and protocols for facilitated peptide mixture analysis and efficient assignment of cross-linked peptide products. In this report, we combine in-source Paterno-Buchi (PB) photo-chemistry with a tandem mass spectrometry approach to selectively address the fragmentation of a tailor-made cross-linking reagent. The PB photochemistry, so far exclusively used for the identification of unsaturation sites in lipids and in lipidomics, is now introduced to the field of chemical cross-linking. Based on trans-3-hexenedioic acid, an olefinic homo bifunctional amine reactive XL reagent was designed and synthesized for this proof-of-principle study. Condensation products of the olefinic reagent with a set of exemplary peptides are used to test the feasibility of the concept. Benzophenone is photochemically reacted in the nano-electrospray ion source and forms oxetane PB reaction products. Subsequent CID-MS triggered retro-PB reaction of the respective isobaric oxetane molecular ions and delivers reliably and predictably two sets of characteristic fragment ions of the cross-linker. Based on these signature ion sets, a straightforward identification of covalently interconnected peptides in complex digests is proposed. Furthermore, CID-MSn experiments of the retro-PB reaction products deliver peptide backbone characteristic fragment ions. Additionally, the olefinic XL reagents exhibit a pronounced robustness upon CID-activation, without previous UV-excitation. These experiments document that a complete backbone fragmentation is possible, while the linker-moiety remains intact. This feature renders the new olefinic linkers switchable between a stable, noncleavable cross-linking mode and an in-source PB cleavable mode.

There is an increasing appreciation that every cell, even of the same type, is different. This complexity, when additionally combined with the variety of different cell types in tissue, is driving the need for spatially resolved omics at the single-cell scale. Rapid advances are being made in genomics and transcriptomics, but progress in metabolomics lags. This is partly because amplification and tagging strategies are not suited to dynamically created metabolite molecules. Mass spectrometry imaging has excellent potential for metabolic imaging. This review summarizes the recent advances in two of these techniques: matrix-assisted laser desorption ionization (MALDI) and secondary ion mass spectrometry (SIMS) and their convergence in subcellular spatial resolution and molecular information. The barriers that have held back progress such as lack of sensitivity and the breakthroughs that have been made including laser-postionization are highlighted as well as the future challenges and opportunities for metabolic imaging at the single-cell scale.

Fermentation broths of Ashbya gossypii from the industrial production of riboflavin emit an intense floral, fruity, and nutty smell. Typical Ehrlich pathway products, such as 2-phenylethan-1-ol and 2-/3-methylbutan-1-ol, were detected in large amounts as well as some intensely smelling saturated and unsaturated lactones, e.g., g-decalactone and g-(Z)-dodec-6-enlactone. An aroma extract dilution analysis identified 2-phenylethan-1-ol and g-(Z)-dodec-6-enlactone as the main contributors to the overall aroma, with flavor dilution factors of 32 768. The position of the double bonds of unsaturated lactones was determined by the Paternò-Büchi reaction, and reference compounds that were not available commercially were synthesized to elucidate the structures of the uncommon lactones. The absolute configuration and enantiomeric excess values of the lactones were determined by converting the lactones to their corresponding Mosher's esters. In addition, the odor impressions and odor thresholds in air were determined.

Metabolomics and lipidomics are new drivers of the omics era as molecular signatures and selected analytes allow phenotypic characterization and serve as biomarkers, respectively. The growing capabilities of untargeted and targeted workflows, which primarily rely on mass spectrometric platforms, enable extensive charting or identification of bioactive metabolites and lipids. Structural annotation of these compounds is key in order to link specific molecular entities to defined biochemical functions or phenotypes. Tandem mass spectrometry (MS), first and foremost collision-induced dissociation (CID), is the method of choice to unveil structural details of metabolites and lipids. But CID fragment ions are often not sufficient to fully characterize analytes. Therefore, recent years have seen a surge in alternative tandem MS methodologies that aim to offer full structural characterization of metabolites and lipids. In this article, principles, capabilities, drawbacks, and first applications of these “advanced tandem mass spectrometry” strategies will be critically reviewed. This includes tandem MS methods that are based on electrons, photons, and ion/molecule, as well as ion/ion reactions, combining tandem MS with concepts from optical spectroscopy and making use of derivatization strategies. In the final sections of this review, the first applications of these methodologies in combination with liquid chromatography or mass spectrometry imaging are highlighted and future perspectives for research in metabolomics and lipidomics are discussed. Graphical abstract

Local lipid variations in tissues are readily revealed with mass spectrometry imaging (MSI) methods, and the resulting lipid distributions serve as bioanalytical signatures to reveal cell- or tissue-specific lipids. Comprehensive MSI lipid mapping requires measurements in both ion polarities. Additionally, structural lipid characterization is necessary to link the lipid structure to lipid function. Whereas some structural elements of lipids are readily derived from high-resolution mass spectrometry (MS) and tandem-MS (MS

Local lipid variations in tissues are readily revealed with mass spectrometry imaging (MSI) methods and resulting lipid distributions serve as bioanalytical signatures to reveal cell- or tissue-specific lipids. Comprehensive MSI lipid mapping requires measurements in both ion polarities. Additionally, structural lipid characterization is necessary to link lipid structure to lipid function. Whereas some structural elements of lipids are readily derived from high-resolution mass spectrometry (MS) and tandem-MS (MSn), the localization of C=C double bonds (DBs) requires specialized fragmentation and/or functionalization methods. In this work, we identify a multifunctional matrix-assisted laser desorption/ionization (MALDI) matrix for spatially-resolved lipidomics investigations that reacts with lipids in Paternò-Büchi (PB) reactions during laser irradiation facilitating DB position assignment and allows dual polarity high-resolution MALDI-MSI and MALDI MS2I studies. By screening twelve compounds for improved ionization efficiency in positive/negative ion mode and PB functionalization yield compared to the previously introduced reactive MALDI matrix benzophenone, benzoylpyridine (BzPy) is identified as the best candidate. The multifunctional character of the new matrix enables DB localization of authentic standards belonging to twelve lipid classes and helps to assign 506/365 lipid features in positive/negative ion mode from mouse cerebellum tissue. The analytical capabilities of BzBy as a multifunctional MALDI-MSI matrix are demonstrated by imaging endogenous and PB-functionalized lipids in mouse kidney sections with 7 µm lateral resolution in both ion modes. Tracking diagnostic lipid DB position fragment ions in mouse pancreas tissue with down to 10 µm pixel size allows to identify islets of Langerhans associated lipid isomer upregulation or depletion.

First results for a new atmospheric-pressure matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging source operating at 213 nm laser wavelength are presented. The activation of analytes in the 213 nm MALDI process at atmospheric pressure was evaluated and compared to results for 337 nm MALDI and electrospray ionization using thermometer molecules. Different sample preparation techniques for nicotinic acid, the matrix with the highest ionization efficiency at 213 nm of all tested matrices, were evaluated and optimized to obtain small crystal sizes, homogenous matrix layer sample coverage, and high ion signal gains. Mass spectrometry imaging experiments of phospholipids in mouse tissue sections in positive- and negative-ion mode with different lateral resolutions and the corresponding pre-/post-mass spectrometry imaging workflows are presented. The use of custom-made objective lenses resulted in sample ablation spot diameters of on average 2.9 μm, allowing mass spectrometry imaging experiments to be performed with 3 μm pixel size without oversampling. The ion source was coupled to an orbital trapping mass spectrometer offering high mass resolution (100.000), high mass accuracy (≤ ±2 ppm), and high sensitivity (single pixel on-tissue tandem MS from 6.6 μm

Lipid oxidation plays a major role in biochemical processes and nutrition. Structural changes during oxidation can lead to alterations of lipid functions. Rancidification and production of secondary lipid messengers are well-known examples for the impact of oxidation on lipid function. Especially lipids with a high degree of unsaturation are prone to oxidize. In order to investigate structural changes of lipids upon oxidation, we here introduce a photochemical Paternò-Büchi functionalization workflow and subsequent mass spectrometric analysis for analysis of unsaturated, oxidized lipids. Results for hydroxylated fatty acids and triglycerides containing isolated and conjugated C[double bond, length as m-dash]C bonds will be presented making use of 3-acetylpyridine as a photochemically active compound. Photochemical derivatization is performed in nano-electrospray emitter tips in 30 s resulting in the formation of oxetanes without inducing light-triggered oxidation of analytes. Collisional-activation of photoproducts facilitates selective cleavage of oxetane moieties. Resulting fragment ions not only allow the determination of C[double bond, length as m-dash]C bond locations for isolated and conjugated C[double bond, length as m-dash]C bonds but also restrict the site of oxidation. By registering the mass shift in some fragment ions of +15.99 Da due to hydroxylation, the oxidized sections of lipids can be identified. In order to demonstrate its analytical robustness, the method is applied to determine the structural impact of non-selective ambient oxidation on fatty acids, triglycerides and complex triglyceride mixtures obtained from Sacha inchi oil.

The location and identity of phospholipids (PLs) within tissues can serve as diagnostic markers for tissue types or diseases. Whereas mass spectrometry imaging (MSI) has emerged as a powerful bioanalytical tool to visualize PL distributions, inferring PL identities from MSI experiments is challenging. Especially, C═C double-bond (DB) positions are not identifiable in most MSI experiments. Herein, we introduce benzophenone (BPh) as a novel reactive matrix for matrix-assisted laser desorption/ionization (MALDI). BPh promotes desorption/ionization and simultaneously serves as derivatization reagent that allows functionalization of unsaturated PLs during the MALDI process via a laser-light driven Paterno-Buchi (PB) reaction without the need for additional equipment. Using BPh, PB product ions of numerous PL classes are readily generated to pinpoint the location of DBs. High lateral resolution MSI results of DB-position isomers are presented, highlighting the capabilities of BPh as a PB-reactive MALDI matrix to potentially unveil the impact of DB-position isomers in PL metabolism.

We report an atmospheric pressure (AP) matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) setup with a lateral resolution of 1.4 μm, a mass resolution greater than 100,000, and accuracy below ±2 p.p.m. We achieved this by coupling a focusing objective with a numerical aperture (NA) of 0.9 at 337 nm and a free working distance of 18 mm in coaxial geometry to an orbitrap mass spectrometer and optimizing the matrix application. We demonstrate improvement in image contrast, lateral resolution, and ion yield per unit area compared with a state-of-the-art commercial MSI source. We show that our setup can be used to detect metabolites, lipids, and small peptides, as well as to perform tandem MS experiments with 1.5-μm2 sampling areas. To showcase these capabilities, we identified subcellular lipid, metabolite, and peptide distributions that differentiate, for example, cilia and oral groove in Paramecium caudatum.

We have developed an effective approach to 1,2-disubstituted diamondoids by palladium(II) acetate catalyzed functionalization of CH bond. Selective mono-arylation of the adamantane framework was achieved using picolylamide as a directing group in yields up to 87 %. Kinetic studies in combination with deuterium labeling experiments, competitive experiments and mass spectrometry contribute to the mechanistic understanding of the arylation process of alkanes with number of CH bonds neighboring the directing group. Triflic anhydride promoted cyclization of the directing group generates imidazo[1,5-a]pyridine derivatives. Acid-mediated removal of the directing group provides access to 2-aryl diamondoid carboxylic acids, which are common precursors for the synthesis of various bioactive compounds (drug candidates).

We describe an atmospheric pressure matrix-assisted laser desorption-ionization mass spectrometry imaging system that uses long-distance laser triangulation on a micrometer scale to simultaneously obtain topographic and molecular information from 3D surfaces. We studied the topographic distribution of compounds on irregular 3D surfaces of plants and parasites, and we imaged nonplanar tissue sections with high lateral resolution, thereby eliminating height-related signal artifacts.

Phosphatidylcholines are the major phospholipid component of most eukaryotic cell membranes. Phosphatidylcholines have been shown to actively participate in regulatory and metabolic processes. Dysfunctional metabolic processes have been linked to human disease and can result in altered phosphatidylcholine structural features, such as permutation of fatty acid connectivity. Assignment and relative quantitation of structural isomers that arise from fatty acid permutation on the phosphatidylcholine backbone, so-called sn-isomers, is difficult with routine tandem mass spectrometry or with liquid chromatography without authentic standards. In this work, we report on the observation that phosphatidylcholines form abundant doubly charged metal ion complexes during electrospray ionization (ESI) and show that these complexes can be used to assign fatty acid moieties, relatively quantify sn-isomers in MS2 experiments, and mass spectrometrically separate phosphatidylcholines from other phospholipid classes in positi...

The ability to control the charge state and ionization efficiency of lipids and hydrocarbons by means of in-source Paternò-Büchi functionalization in nano-electrospray ionization mass spectrometry experiments is investigated. Ultraviolet light irradiation of acetylpyridine filled nano-electrospray emitter tips, containing unsaturated analytes, generates protonated lipid and hydrocarbon ions. Comparison of reaction yields and fragment ion abundances of functionalized unsaturated fatty acids indicate that acetylpyridine Paternò-Büchi functionalization allows to readily detect fatty acids and determine double bond positions, but fragmentation efficiency and reactivity depend on double bond position and varies between different acetylpyridine isomers. Results for methyl oleate and olefins suggest that fragment ion abundances of unsaturated compounds depend on interactions between acetylpyridine and nearby functional groups. Paternò-Büchi functionalization with acetylpyridine was used to detect and assign double bond positions of mono- and polyunsaturated fatty acid, cholesterol ester, triglyceride, and hydrocarbon standards with ion abundances that are up to 631 times higher than abundances of the same compounds prior Paternò-Büchi reaction. To demonstrate the scope and analytical robustness of the newly developed method, free fatty acids in mouse brain as well as male Schistosoma mansoni extracts and hydrocarbons in an olefin mixture are investigated. For this complex set of analytes, charging and charge switching using acetylpyridine Paternò-Büchi functionalization enable double bond position assignment and relative quantification in positive ion mode. Graphical Abstract.

RATIONALE Neurosteroids are important signalling molecules that modulate neuronal activity. Their low concentrations and low volatility make neurosteroid detection and quantification by ambient mass spectrometry challenging. Here we develop a reactive low-temperature plasma mass spectrometry (LTP-MS) method and demonstrate its potential for fast screening and quantification of neurosteroids in mouse brain. METHODS Ketone-based neurosteroids were analysed with the LTP-MS method. The plasma of the LTP was heated in order to improve the desorption efficiency of low-volatility neurosteroids. Methylamine with a concentration of 500 ppbv was employed as the reactive reagent. Neurosteroids in mouse brain tissue extracts were detected in 70 s with mass errors less than ±3 ppm due to coupling of the ion source with a high-performance mass spectrometer. RESULTS Reaction between neurosteroids and methylamine, seeded into the LTP gas stream, resulted in the formation of protonated methylamine-neurosteroid adducts with 5- to 100-fold abundances, compared to [M + H]+ ions detected in non-reactive LTP-MS. The lowest detectable concentrations of neurosteroid standards were in the range of ng/mL. Concentrations of neurosteroids in male and female mouse brain extracts as determined with reactive LTP-MS were on the level of ng/g, comparable to results obtained with high-performance liquid chromatography-tandem mass spectrometry. CONCLUSIONS The developed reactive LTP-MS is capable of providing sensitive identification and quantification of ketone-based neurosteroids in mouse brain extracts with minimal sample treatment, and showcases the potential of reactive LTP-MS as a tool for fast screening of neurosteroid levels in brain.

Structures of deprotonated Cys, Asp, Glu, Phe, Pro, His homo dimers as well as [2Cys − 3H]−, [Asp + Glu − H]− and [2Glu − 2H + Na]− are investigated with infrared multiple-photon dissociation (IRMPD) spectroscopy between 650 and 1850 cm−1 and theory. The IRMPD spectra of all investigated complexes but [2His − H]−, [2Phe − H]− and [2Pro − H]− indicate that the structures consist of a neutral non-zwitterionic (NZ) and a deprotonated form of the amino acids. In contrast, the spectrum of [2His − H]− is complex and indicates the presence of multiple isomers and/or interactions between His and [His − H]−, so that its structure differs from that of the other deprotonated amino acid dimers. For [2Phe − H]− and especially for [2Pro − H]−, some IRMPD bands can only be explained by the presence of salt bridge (SB) structures in the dimer in which a deprotonated amino acid interacts with a zwitterionic neutral amino acid. Computational results indicate that SB structures are lower in energy at 298 K than corresponding NZ structures for neutral-anion complexes in which SB formation is not disrupted by amino acid side chains or conformational constraints, such as in [2Glu − H]− and [2Cys − 3H]− for which NZ structures are most consistent with experimental results. For deprotonated amino acid dimers in which these interfering interactions are absent, such as in [2Phe − H]− and [2Pro − H]−, the higher number of hydrogen bonds in SB compared to NZ structures stabilize the formation of zwitterionic neutral amino acids and consequently SB structures in agreement with results from IRMPD spectroscopy. These results suggest that SB structures likely occur in deprotonated peptide or protein ions at hydrophobic sites, such as protein–protein interfaces or in the interior of proteins, where interfering functional groups will not disrupt SB formation

The effect of double bond functionalisation for selective double bond localisation by ultraviolet photodissociation of phosphatidylcholines is investigated. Paterno–Buchi reactions in nanoESI emitter tips enable attachment of acetophenone to double bonds of unsaturated phosphatidylcholines after 100 s of 254 nm light irradiation with about 50–80% reaction yield. Functionalized phosphatidylcholines dissociate upon 266 nm irradiation yielding double bond selective fragment ions in contrast to results for ultraviolet photodissociation of unmodified lipids. Ultraviolet photodissociation of Paterno–Buchi modified lipids results in a selectivity increase of up to 2.2 towards double bond localisation compared collision-induced dissociation experiments. Double bond localisation is also possible with ultraviolet photodissociation when alkali metal ion attachment to Paterno–Buchi modified phosphatidylcholines occurs in contrast to classic collision-induced dissociation experiments. The developed methodology is used to differentiate lipid double bond isomers and applied to phosphatidylcholines from egg yolk to identify 15 phosphatidylcholines. Results from this study demonstrate that locally depositing energy in close vicinity to cleavable bonds via ultraviolet photodissociation can result in increased dissociation selectivity. This method can help to disentangle contributions from different structural elements in complex tandem mass spectra of lipids and aid to the structural characterization of phospholipids in a “top-down” approach.

Ultraviolet photodissociation tandem mass spectrometry is a powerful tool to investigate the structure of biomolecules, due to its ability to generate rich fragmentation patterns or bond selective cleavage, as a function of used laser wavelength, laser fluence, dose (number of accumulated laser pulses), and available chromophores. Herein, we report first results obtained with a newly developed two-wavelength (266 nm and 213 nm) ultraviolet photodissociation setup coupled to a Fourier-transform ion cyclotron resonance mass spectrometer. Photoproduct yields for protonated 3-iodo-l-tyrosine were up to ∼75%. Dose and fluence dependent measurements for protonated 3-iodo-l-tyrosine, doubly charged protonated bradykinin and Fe(II) attached to 1,2-dioleoyl-sn-glycero-3-phosphocholine reveal that the ultraviolet photodissociation mechanism for photoproduct formation qualitatively differs between these model systems. Three derived photodissociation models were used to interpret the experimental results and show that while protonated 3-iodo-l-tyrosine and Fe(II) attached to 1,2-dioleoyl-sn-glycero-3-phosphocholine most likely dissociates via a single-photon process, fragmentation of doubly charged bradykinin ions was found to be most consistent with sequential two-photon dissociation (213 nm). The introduced dissociation models present an easy means to study the mechanism of ultraviolet photodissociation processes for a variety of analytes without prior knowledge of their photochemistry or to optimize experimental conditions by adjusting laser fluence or number of laser pulses.

The novel surface mode of the Birmingham Cluster Genetic Algorithm (S-BCGA) is employed for the global optimisation of noble metal tetramers upon an MgO (100) substrate at the GGA-DFT level of theory. The effect of element identity and alloying in surface-bound neutral subnanometre clusters is determined by energetic comparison between all compositions of PdnAg(4−n) and PdnPt(4−n). While the binding strengths to the surface increase in the order Pt > Pd > Ag, the excess energy profiles suggest a preference for mixed clusters for both cases. The binding of CO is also modelled, showing that the adsorption site can be predicted solely by electrophilicity. Comparison to CO binding on a single metal atom shows a reversal of the 5σ–d activation process for clusters, weakening the cluster–surface interaction on CO adsorption. Charge localisation determines homotop, CO binding and surface site preferences. The electronic behaviour, which is intermediate between molecular and metallic particles allows for tunable features in the subnanometre size range.

Over the past decade, there has been a significant growth in the development and application of methods for performing global optimization (GO) of cluster and nanoparticle structures using first-principles electronic structure methods coupled to sophisticated search algorithms. This has in part been driven by the desire to avoid the use of empirical potentials (EPs), especially in cases where no reliable potentials exist to guide the search toward reasonable regions of configuration space. This has been facilitated by improvements in the reliability of the search algorithms, increased efficiency of the electronic structure methods, and the development of faster, multiprocessor high-performance computing architectures. In this review, we give a brief overview of GO algorithms, though concentrating mainly on genetic algorithm and basin hopping techniques, first in combination with EPs. The major part of the review then deals with details of the implementation and application of these search methods to allow exploration for global minimum cluster structures directly using electronic structure methods and, in particular, density functional theory. Example applications are presented, ranging from isolated monometallic and bimetallic clusters to molecular clusters and ligated and surface supported metal clusters. Finally, some possible future developments are highlighted. © 2013 Wiley Periodicals, Inc.

Sequential water binding enthalpies for aqueous nanodrops containing 20–500 water molecules and a 1+ to 3+ ion are reported., Sequential water molecule binding enthalpies, ΔH n,n–1, are important for a detailed understanding of competitive interactions between ions, water and solute molecules, and how these interactions affect physical properties of ion-containing nanodrops that are important in aerosol chemistry. Water molecule binding enthalpies have been measured for small clusters of many different ions, but these values for ion-containing nanodrops containing more than 20 water molecules are scarce. Here, ΔH n,n–1 values are deduced from high-precision ultraviolet photodissociation (UVPD) measurements as a function of ion identity, charge state and cluster size between 20–500 water molecules and for ions with +1, +2 and +3 charges. The ΔH n,n–1 values are obtained from the number of water molecules lost upon photoexcitation at a known wavelength, and modeling of the release of energy into the translational, rotational and vibrational motions of the products. The ΔH n,n–1 values range from 36.82 to 50.21 kJ mol–1. For clusters containing more than ∼250 water molecules, the binding enthalpies are between the bulk heat of vaporization (44.8 kJ mol–1) and the sublimation enthalpy of bulk ice (51.0 kJ mol–1). These values depend on ion charge state for clusters with fewer than 150 water molecules, but there is a negligible dependence at larger size. There is a minimum in the ΔH n,n–1 values that depends on the cluster size and ion charge state, which can be attributed to the competing effects of ion solvation and surface energy. The experimental ΔH n,n–1 values can be fit to the Thomson liquid drop model (TLDM) using bulk ice parameters. By optimizing the surface tension and temperature change of the logarithmic partial pressure for the TLDM, the experimental sequential water molecule binding enthalpies can be fit with an accuracy of ±3.3 kJ mol–1 over the entire range of cluster sizes.

A genetic algorithm (GA) coupled with density functional theory (DFT) calculations is used to perform global optimisations for all compositions of 8-atom Au–Ag bimetallic clusters. The performance of this novel GA-DFT approach for bimetallic nanoparticles is tested for structures reported in the literature. New global minimum structures for various compositions are predicted and the 2D–3D transition is located. Results are explained with the aid of an analysis of the electronic density of states. The chemical ordering of the predicted lowest energy isomers are explained via a detailed analysis of the charge separation and mixing energies of the bimetallic clusters. Finally, dielectric properties are computed and the composition and dimensionality dependence of the electronic polarizability and dipole moment is discussed, enabling predictions to be made for future electric beam deflection experiments.

Photo-ionization mass spectrometry and electric beam deflection experiments were used to study isolated Sn(M)Pb(N) clusters (7or=N + Mor= 13 for tin-rich clusters, 7or=N + Mor= 15 for lead-rich clusters) in a molecular beam apparatus. The observed mass spectra reveal a broad abundance distribution of the bimetallic clusters in which all possible cluster compositions can be identified within the investigated size ranges. Comparison of the relative cluster intensities between pure tin or lead clusters (Sn(N+M) and Pb(N+M)) and mixed Sn(M)Pb(N) clusters indicate quite similar relative abundance distributions which can be smoothly shifted from one to the other extreme by changing the composition. The mass spectroscopic findings could be explained by assuming a substitution "alloy" formation in the Sn(M)Pb(N) cluster system. In combination, the dielectric properties were determined by passing the bimetallic clusters through an inhomogeneous electric field. The observed polarizabilities are significantly increased for most of the bimetallic clusters. This can be explained in an adiabatic polarization model by the presence of permanent electric dipole moments. These observations demonstrate how the electronic properties are not only crucially influenced by the cluster size but also by the composition of this nanoalloy model system. In addition to the enhanced polarizability, most of the measured beam profiles for tin-rich clusters show detectable beam broadenings due to the permanent dipole moments, in contrast to lead-rich clusters which possess considerable smaller dipole moments. Molecular dynamic simulations of the measured beam profile for Sn(6)Pb(1) taking theoretically calculated isomeric structures and dipole moments into account yields no completely satisfying outcome. Therefore we discuss possible reasons for the discrepancy between experimental and theoretical results.

Photocontrol of a piperidine's Brønsted basicity was achieved by incorporation of a bulky azobenzene group and could be translated into pronounced reactivity differences between ON- and OFF-states in general base catalysis. This enabled successful photomodulation of the catalyst's activity in the nitroaldol reaction (Henry reaction). A modular synthetic route to the photoswitchable catalysts was developed and allowed for preparation and characterization of three azobenzene-derived bases as well as one stilbene-derived base. Solid-state structures obtained by X-ray crystal structure analysis confirmed efficient blocking of the active site in the E isomer representing the OFF-states, whereas a freely accessible active site was revealed for a representative Z isomer in the crystal. To correlate structure with reactivity of the catalysts, conformational dynamics were thoroughly studied in solution by NMR spectroscopy, taking advantage of residual dipolar couplings (RDCs), in combination with comprehensive DFT computational investigations of conformations and proton affinities.

Hydration of PbNO3(+) and SrNO3(+) with up to 30 water molecules was investigated with infrared photodissociation (IRPD) spectroscopy and with theory. These ions are the same size, yet the IRPD spectra of these ion pairs for n = 2-8 are significantly different. Bands in the bonded O-H region (∼3000-3550 cm(-1)) indicate that the onset of a second hydration shell begins at n = 5 for PbNO3(+) and n = 6 for SrNO3(+). Spectra for [PbNO3](+)(H2O)2-5 and [SrNO3](+)(H2O)3-6 indicate that the structures of clusters with Pb(ii) are hemidirected with a void in the coordinate sphere. A natural bond orbital analysis of [PbNO3](+)(H2O)5 indicates that the anisotropic solvation of the ion is due to a region of asymmetric electron density on Pb(ii) that can be explained by charge transfer from the nitrate and water ligands into unoccupied p-orbitals on Pb(ii). There are differences in the IRPD spectra of PbNO3(+) and SrNO3(+) with up to 25 water molecules attached. IR intensity in the bonded O-H region is blue-shifted by ∼50 cm(-1) in nanodrops containing SrNO3(+) compared to those containing PbNO3(+), indicative of a greater perturbation of the water H-bond network by strontium. The free O-H stretches of surface water molecules in nanodrops containing 10, 15, 20, and 25 water molecules are red-shifted by ∼3-8 cm(-1) for PbNO3(+) compared to those for SrNO3(+), consistent with more charge transfer between water molecules and Pb(ii). These results demonstrate that the different electronic structure of these ions significantly influences how they are solvated.

Hydration of gaseous guanidinium (Gdm+) with up to 100 water molecules attached was investigated using infrared photodissociation spectroscopy in the hydrogen stretch region between 2900 and 3800 cm-1. Comparisons to IR spectra of low-energy computed structures indicate that at small cluster size, water interacts strongly with Gdm+ with three inner shell water molecules each accepting two hydrogen bonds from adjacent NH2 groups in Gdm+. Comparisons to results for tetramethylammonium (TMA+) and Na+ enable structural information for larger clusters to be obtained. The similarity in the bonded OH region for Gdm(H2O)20+vs. Gdm(H2O)100+ and the similarity in the bonded OH regions between Gdm+ and TMA+ but not Na+ for clusters with

Structures and reactivities of gaseous Fe(CN)(6)(3-)(H(2)O)n were investigated using infrared photodissociation (IRPD) kinetics, spectroscopy, and computational chemistry in order to gain insights into how water stabilizes highly charged anions. Fe(CN)(6)(3-)(H(2)O)(8) is the smallest hydrated cluster produced by electrospray ionization, and blackbody infrared dissociation of this ion results in loss of an electron and formation of smaller dianion clusters. Fe(CN)(6)(3-)(H(2)O)(7) is produced by the higher activation conditions of IRPD, and this ion dissociates both by loss of an electron and by loss of a water molecule. Comparisons of IRPD spectra to those of computed low-energy structures for Fe(CN)(6)(3-)(H(2)O)(8) indicate that water molecules either form two hydrogen bonds to the trianion or form one hydrogen bond to the ion and one to another water molecule. Magic numbers are observed for Fe(CN)(6)(3-)(H(2)O)n for n between 58 and 60, and the IRPD spectrum of the n = 60 cluster shows stronger water molecule hydrogen-bonding than that of the n = 61 cluster, consistent with the significantly higher stability of the former. Remarkably, neither cluster has a band corresponding to a free O-H stretch, and this band is not observed for clusters until n ≥ 70, indicating that this trianion significantly affects the hydrogen-bonding network of water molecules well beyond the second and even third solvation shells. These results provide new insights into the role of water in stabilizing high-valency anions and how these ions can pattern the structure of water even at long distances.

The formation of a salt bridge in deprotonated glycine dimer anions in a solvent-free environment is investigated using both infrared multiple photon dissociation spectroscopy between 600 and 1800 cm(-1) and theory. The zwitterionic and nonzwitterionic forms of glycine in this complex are computed to be nearly iso-energetic, yet predominantly the zwitterionic form is observed experimentally. The zwitterion stability is attributed to both the Coulombic attraction and the high stabilization from intramolecular hydrogen bonding that drives the energetic cost of proton transfer in a solvent free environment. These results show that there is a fine balance between the stabilities of these two forms of the anion. Elucidating the role of intrinsic factors, such as hydrogen bonding, can lead to a better understanding of the stabilities of salt bridges in the interiors of large proteins or at protein interfaces.

The structures of hydrated guanidinium, Gdm(+)(H2O)n, where n = 1-5, were investigated with infrared photodissociation spectroscopy and with theory. The spectral bands in the free O-H (∼3600-3800 cm(-1)) and free N-H (∼3500-3600 cm(-1)) regions indicate that, for n between 1 and 3, water molecules bind between the NH2 groups in the plane of the ion forming one hydrogen bond with each amino group. This hydration structure differs from Gdm(+) in solution, where molecular dynamics simulations suggest that water molecules form linear H-bonds with the amino groups, likely a result of additional water-water interactions in solution that compete with the water-guanidinium interactions. At n = 4, changes in the free O-H and bonded O-H (∼3000-3500 cm(-1)) regions indicate water-water H-bonding and thus the onset of a second hydration shell. An inner shell coordination number of n = 3 is remarkably small for a monovalent cation. For Gdm(+)(H2O)5, the additional water molecule forms hydrogen bonds to other water molecules and not to the ion. These results indicate that Gdm(+) is weakly hydrated, and interactions with water molecules occur in the plane of the ion. This study offers the first experimental assignment of structures for small hydrates of Gdm(+), which provide insights into the unusual physicochemical properties of this ion.

After having explained the experimental realization of beam deflection experiments in Chap. 2 , this chapter will introduce various interpretation schemes of the experimental results. Depending on the experimental conditions and the studied system either the rigid rotor model or the floppy cluster assumption must be applied. For the rigid rotor model perturbation theory methods, classical and quantum chemical simulations are discussed and their performance is compared. The latter two methods require a model of the geometric structure of the cluster and the corresponding dielectric properties. Therefore, a very brief introduction of unbiased structure search routines and quantum chemical computations is given. For floppy or thermally activated clusters the simple Langevin-Debye model is introduced while a few more sophisticated methods are discussed at the end of the chapter.