Investigation of the laser fluence and wavelength dependence in surface-assisted laser desorption/ionization mass spectrometry using gold nanoparticles.

RATIONALE: Surface-assisted laser desorption/ionization (SALDI) mass spectrometry (MS) builds on the use of nanostructured surfaces (e.g., coatings of colloidal nanoparticles) to promote analyte desorption and ionization. The SALDI process is believed to occur mainly through thermal processes, resulting from heating of the nanosubstrate upon absorption of the photon energy, and by assisting ionization steps. Mostly due to the accessibility of the respective hardware, the majority of SALDI-MS studies use standard laser wavelengths for MALDI (i.e., 337 or 355 nm), even though peak absorption of the SALDI nanosubstrate might completely differ from these values. METHODS: Here, we investigated the wavelength dependence in SALDI-MS to determine if wavelength adjustment would be beneficial, and to provide new experimental data for a better understanding of the SALDI mechanism. To this end, gold nanoparticles (AuNPs) sprayed onto microscope glass slides were employed as SALDI nanosubstrates and L-arginine as a model analyte. In addition, we used 2,5-dihydroxyacetophenone (2,5-DHAP) for classical MALDI-MS using the same experimental setup. Arginine ion signals were recorded as a function of laser wavelength and laser fluence. Mass spectra were acquired in the wavelength range between 310 and 630 nm, including the absorption maximum of the sprayed AuNPs around 550 nm and that of 2,5-DHAP around 380 nm. RESULTS: Laser fluence thresholds for the generation of arginine ions were found to be dependent on the laser wavelength and to inversely correlate with the absorbance profiles of the deposited AuNPs and 2,5-DHAP, respectively. Very differently to MALDI, in SALDI ionization efficiency was found to strictly linearly decrease with increasing laser wavelength. CONCLUSIONS: Our results, therefore, corroborate the general assumption that material ejection in SALDI-MS is mainly driven by thermal processes in the low laser fluence range and add new evidence that the ionization process is directly influenced by photon energy when AuNPs are employed as nanosubstrates.

Large-Scale Screening of Pharmaceutical Compounds to Explore the Application Space of On-Tissue MALDI and MALDI-2 Mass Spectrometry.

The successful application of matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI) in pharmaceutical research is strongly dependent on the detection of the drug of interest at physiologically relevant concentrations. Here we explored how insufficient sensitivity due to low ionization efficiency and/or the interaction of the drug molecule with the local biochemical environment of the tissue can be mitigated for many compound classes using the recently introduced MALDI-MSI coupled with laser-induced postionization, known as MALDI-2-MSI. Leveraging a MALDI-MSI screen of about 1,200 medicines/drug-like compounds from a broad range of medicinal application areas, we demonstrate a significant improvement in drug detection and the degree of sensitivity uplift by using MALDI-2 versus traditional MALDI. Our evaluation was made under simulated imaging conditions using liver homogenate sections as substrate, onto which the compounds were spotted to mimic biological conditions to the first order. To enable an evaluable detection by both MALDI and MALDI-2 for the majority of employed compounds, we spotted 1 µL of a 10 mM solution using a spotting robot and performed our experiments with a Bruker timsTOF fleX MALDI-2 instrument in both positive and negative ion modes. Specifically, we demonstrate using a large cohort of drug-like compounds that ∼60% of the tested compounds showed a more than 10-fold increase in signal intensity and ∼16% showed a more than 100-fold increase upon use of MALDI-2 postionization. Such increases in sensitivity could help advance pharmaceutical MALDI-MSI applications toward the single-cell level.

Visualization of metabolites and microbes at high spatial resolution using MALDI mass spectrometry imaging and in situ fluorescence labeling.

Label-free molecular imaging techniques such as matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI) enable the direct and simultaneous mapping of hundreds of different metabolites in thin sections of biological tissues. However, in host-microbe interactions it remains challenging to localize microbes and to assign metabolites to the host versus members of the microbiome. We therefore developed a correlative imaging approach combining MALDI-MSI with fluorescence in situ hybridization (FISH) on the same section to identify and localize microbial cells. Here, we detail metaFISH as a robust and easy method for assigning the spatial distribution of metabolites to microbiome members based on imaging of nucleic acid probes, down to single-cell resolution. We describe the steps required for tissue preparation, on-tissue hybridization, fluorescence microscopy, data integration into a correlative image dataset, matrix application and MSI data acquisition. Using metaFISH, we map hundreds of metabolites and several microbial species to the micrometer scale on a single tissue section. For example, intra- and extracellular bacteria, host cells and their associated metabolites can be localized in animal tissues, revealing their complex metabolic interactions. We explain how we identify low-abundance bacterial infection sites as regions of interest for high-resolution MSI analysis, guiding the user to a trade-off between metabolite signal intensities and fluorescence signals. MetaFISH is suitable for a broad range of users from environmental microbiologists to clinical scientists. The protocol requires ~2 work days.

Visualization of Differential Cardiolipin Profiles in Murine Retinal Cell Layers by High-Resolution MALDI Mass Spectrometry Imaging.

The precise fatty acyl chain configuration of cardiolipin (CL), a tetrameric mitochondrial-specific membrane lipid, exhibits dependence on cell and tissue types. A powerful method to map CL profiles in tissue sections in a spatially resolved manner is matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI). To build on and explore this potential, we employed a quadrupole time-of-flight mass spectrometer along with optimized sample preparation protocols. We imaged the CL profiles of individual murine retinal cell layers at a pixel size of 10 µm. In combination with tandem MS, we obtained detailed insights into the CL composition of individual retinal cell layers. In particular, we found differential expression of the polyunsaturated fatty acids (PUFA) linoleic, arachidonic, and docosahexaenoic acids. PUFAs are prone to peroxidation and hence regarded as critical factors in development and progression of retinal pathologies, such as age-related macular degeneration (AMD). The ability of MALDI-MSI to provide cues on the CL composition in neuronal tissue with close to single-cell resolution can provide important insights into retinal physiology in health and disease.

Mass spectrometry imaging to explore molecular heterogeneity in cell culture.

Molecular analysis on the single-cell level represents a rapidly growing field in the life sciences. While bulk analysis from a pool of cells provides a general molecular profile, it is blind to heterogeneities between individual cells. This heterogeneity, however, is an inherent property of every cell population. Its analysis is fundamental to understanding the development, function, and role of specific cells of the same genotype that display different phenotypical properties. Single-cell mass spectrometry (MS) aims to provide broad molecular information for a significantly large number of cells to help decipher cellular heterogeneity using statistical analysis. Here, we present a sensitive approach to single-cell MS based on high-resolution MALDI-2-MS imaging in combination with MALDI-compatible staining and use of optical microscopy. Our approach allowed analyzing large amounts of unperturbed cells directly from the growth chamber. Confident coregistration of both modalities enabled a reliable compilation of single-cell mass spectra and a straightforward inclusion of optical as well as mass spectrometric features in the interpretation of data. The resulting multimodal datasets permit the use of various statistical methods like machine learning-driven classification and multivariate analysis based on molecular profile and establish a direct connection of MS data with microscopy information of individual cells. Displaying data in the form of histograms for individual signal intensities helps to investigate heterogeneous expression of specific lipids within the cell culture and to identify subpopulations intuitively. Ultimately, t-MALDI-2-MSI measurements at 2-µm pixel sizes deliver a glimpse of intracellular lipid distributions and reveal molecular profiles for subcellular domains.

Single-Photon-Induced Post-Ionization to Boost Ion Yields in MALDI Mass Spectrometry Imaging.

Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) is a rapidly growing method in the life sciences. However, for many analyte classes, its sensitivity is limited due to poor ionization efficiencies. To mitigate this problem, we here introduce a novel post-ionization scheme based on single-photon induced chemical ionization using pulsed RF-Kr lamps. The fine-vacuum conditions of a dual ion-funnel ion source effectively thermalize the evolving MALDI plume and enable ample gas-phase reactions. Injected chemical dopants crucially support fragment-less ionization to [M+H]+ /[M-H]- species. Based on this interplay, numerous glycerophospho-, sphingo-, and further lipids, registered from mammalian tissue sections, were boosted by up to three orders of magnitude, similar to results obtained with laser-based post-ionization (MALDI-2). Experiments with deuterated matrix and dopant, however, indicated complex chemical ionization pathways different from MALDI-2.

Mass Spectrometry Imaging Techniques Enabling Visualization of Lipid Isomers in Biological Tissues.

This Feature focuses on a review of recent developments in mass spectrometry imaging (MSI) of lipid isomers in biological tissues. The tandem MS techniques utilizing online and offline chemical derivatization procedures, ion activation techniques such as ozone-induced dissociation (OzID), ultraviolet photodissociation (UVPD), or electron-induced dissociation (EID), and other techniques such as coupling of ion mobility with MSI are discussed. The importance of resolving lipid isomers in diseases is highlighted.

Neglected Very Long-Chain Hydrocarbons and the Incorporation of Body Surface Area Metrics Reveal Novel Perspectives for Cuticular Profile Analysis in Insects.

Most of our knowledge on insect cuticular hydrocarbons (CHCs) stems from analytical techniques based on gas-chromatography coupled with mass spectrometry (GC-MS). However, this method has its limits under standard conditions, particularly in detecting compounds beyond a chain length of around C40. Here, we compare the CHC chain length range detectable by GC-MS with the range assessed by silver-assisted laser desorption/ionization mass spectrometry (Ag-LDI-MS), a novel and rarely applied technique on insect CHCs, in seven species of the order Blattodea. For all tested species, we unveiled a considerable range of very long-chain CHCs up to C58, which are not detectable by standard GC-MS technology. This indicates that general studies on insect CHCs may frequently miss compounds in this range, and we encourage future studies to implement analytical techniques extending the conventionally accessed chain length range. Furthermore, we incorporate 3D scanned insect body surface areas as an additional factor for the comparative quantification of extracted CHC amounts between our study species. CHC quantity distributions differed considerably when adjusted for body surface areas as opposed to directly assessing extracted CHC amounts, suggesting that a more accurate evaluation of relative CHC quantities can be achieved by taking body surface areas into account.

Effect of the Laser Pulse Width in MALDI-2: A Comparative Study of Picosecond versus Nanosecond Wide Pulses for Laser Postionization.

MALDI-2 is a recently introduced technique for postionization (PI) in matrix-assisted laser desorption/ionization (MALDI). It is based on an initial photoionization of neutrally desorbed matrix molecules and subsequent charge-transfer reactions in a fine vacuum or atmospheric pressure ion source. MALDI-2 significantly increases the ion yields for numerous classes of analytes, including lipids, glycans, and a range of pharmaceuticals. To obtain insights into the ionization mechanisms underlying the primary step of PI in MALDI-2, we here conducted a set of experiments with two lasers at 266 nm wavelength and pulse durations of 28 ps and 6 ns, respectively, on a modified orthogonal-extracting time-of-flight mass spectrometer (QTOF, Synapt). 2,5-Dihydroxybenzoic acid (DHB) and 2,5-dihydroxyacetophenone (DHAP) were investigated as MALDI matrices in the positive-ion mode with standardized lipid samples. Analyte- and matrix-derived ion signals were recorded as a function of PI laser pulse energies. The ion signal intensity displays a quadratic dependency on PI-laser pulse energy for low to moderate intensities of up to ∼107 W/cm2. This behavior suggests the involvement of resonance enhanced two-photon ionization (REMPI) of neutral matrix molecules in the ionization pathways. Comparing nanosecond and picosecond pulses at the same PI laser pulse energy, higher photon density produced by the shorter pulses generally produced sizably higher ion signal intensities, also corroborating an involvement of REMPI-like processes. Based on a theoretical description of the MALDI-2 process derived from prevalent REMPI theory, comparative measurements allow us to determine the lifetime of the excited states of the employed matrices. Resulting values for both matrices are in good agreement with the literature and corroborate the REMPI-based approach.

MALDI-2 and t-MALDI-2 Mass Spectrometry Imaging.

Matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI) combined with laser-induced postionization for MALDI-2 enables the simultaneous registration of numerous classes of small molecules (e.g., secondary metabolites including sterols) as well as phospholipids, glycolipids, and glycans from tissue sections and from cell cultures with strongly boosted ion yields. Here, we describe methodological aspects that are key for optimizing the analytical sensitivity and spatial resolution of a MALDI-2 imaging experiment. We will include both top-illumination MALDI-2 as well as the recently introduced transmission (t-) mode MALDI-2 approach.

Infrared MALDI Mass Spectrometry with Laser-Induced Postionization for Imaging of Bacterial Colonies.

Ultraviolet matrix-assisted laser desorption ionization mass spectrometry imaging (UV-MALDI-MSI) is a powerful tool to visualize bacterial metabolites in microbial colonies and in biofilms. However, a challenge for the method is the efficient extraction of analytes from deeper within the bacterial colonies and from the cytoplasm of individual cells during the matrix coating step. Here, we used a pulsed infrared (IR) laser of 2.94 µm wavelength to disrupt and ablate bacterial cells without a prior coating with a MALDI matrix. Instead, tissue water or, in some experiments, in addition a small amount of glycerol was exploited for the deposition of the IR laser energy and for supporting the ionization of the analytes. Compared to water, glycerol exhibits a lower vapor pressure, which prolonged the available measurement time window within an MSI experiment. Mass spectra were acquired with a hybrid Synapt G2-S HDMS instrument at a pixel size of 120 µm. A frequency-quadrupled q-switched Nd:YAG laser with 266 nm wavelength served for laser-induced postionization (MALDI-2). In this way, the ion abundances of numerous small molecules such as nucleobases, 2-alkyl-quinolones, a prominent class of Pseudomonas aeruginosa signaling molecules involved in one of the three quorum-sensing pathways, and also the signals of various bacterial phospholipids were boosted, partially by orders of magnitude. We analyzed single and cocultured colonies of Gram-negative P. aeruginosa and of Gram-positive Bacillus subtilis and Staphylococcus aureus as exemplary bacterial systems. To enable a rapid (within 5 s) MSI-compatible steam inactivation in a custom-made autoclave filled with hot water steam, bacterial cultures were grown on porous polyamide membranes. Compared to a UV-MALDI-2-MS measurement of the same systems, mass spectra with a reduced low mass background were generally generated. This resulted in the unequivocal detection of numerous metabolites only with the IR laser. In a fundamental part of our study, and to optimize the IR-MALDI-2 approach for the highest analytical sensitivity, we characterized the expansion dynamics of the particle plume as generated by the IR laser. Here, we recorded the total ion count and the intensities of selected signals registered from P. aeruginosa samples as a function of the interlaser delay and buffer gas pressure in the ion source. The data revealed that the IR-MALDI-2 ion signals are primarily generated from slow particles having mean velocities of ∼10 m/s. Interestingly, two different pressure/delay time regimes of the optimized ionization efficiency for phospholipids and smaller metabolites, respectively, were revealed, a result pointing to yet-unknown convoluted reaction cascades. The described IR-MALDI-2 method could be a helpful new tool for a microbial mass spectrometry imaging of small molecules requiring little sample preparation.

Transmission-Mode MALDI Mass Spectrometry Imaging of Single Cells: Optimizing Sample Preparation Protocols.

Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) makes it possible to simultaneously visualize the spatial distribution of dozens to hundreds of different biomolecules (e.g., phospho- and glycolipids) in tissue sections and in cell cultures. The implementation of novel desorption and (post-)ionization techniques has recently pushed the pixel size of this imaging technique to the low micrometer scale and below and thus to a cellular and potentially sub-cellular level. However, to fully exploit this potential for cell biology and biomedicine, sample preparation becomes highly demanding. Here, we investigated the effect of several key parameters on the quality of the sample preparation and achievable spatial resolution, that include the washing, drying, chemical fixation, and matrix coating steps. The incubation of cells with formalin for about 5 min in combination with isotonic washing and mild drying produced a robust protocol that largely preserved not only cell morphologies, but also the molecular integrities of amine group-containing cell membrane phospholipids (phosphatidylethanolamines and -serines). A disadvantage of the chemical fixation is an increased permeabilization of cell membranes, resulting in leakage of cytosolic compounds. We demonstrate the pros and cons of the protocols with four model cell lines, cultured directly on indium tin oxide (ITO)-coated glass slides. Transmission (t-)mode MALDI-2-MSI enabled on a Q Exactive plus Orbitrap mass spectrometer was used to analyze the cultures at a pixel size of 2 µm. Phase contrast light microscopy and scanning electron microscopy were used as complementary methods. The protocols described could prove to be an important contribution to the advancement of single-cell MALDI imaging, especially for the characterization of cell-to-cell heterogeneities at a molecular level.

Spatial distribution of isobaric androgens in target tissues using chemical derivatization and MALDI-2 on a trapped ion mobility quadrupole time-of-flight instrument.

Prostate cancer is initially treated via androgen deprivation therapy (ADT), a highly successful treatment in the initial pursuit of tumour regression, but commonly restricted by the eventual emergence of a more lethal 'castrate resistant' (CRPC) form of the disease. Intracrine pathways that utilize dehydroepiandrosterone (DHEA) or other circulatory precursor steroids are thought to generate relevant levels of growth-stimulating androgens such as testosterone (T) and dihydrotestosterone (DHT). Decoding this tissue-specific metabolic pathway is key for the development of novel therapeutic treatments. Mass spectrometry imaging (MSI) is an analytical technique that allows the visualization of the distribution of numerous classes of biomolecules within tissue sections. The analysis of androgens by liquid chromatography mass spectrometry (LC/MS)-based methods however presents a challenge due to their generally poor ionization efficiency and low physiological endogenous levels. In MSI, on-tissue chemical derivatization (OTCD) has enabled the limits of steroids to be imaged within tissues to be pushed by overcoming poor ionization performance. However, isobaric interference of key androgen derivatives such as T and DHEA can severely hamper studying the intracrinology in several diseases. Here, we have evaluated the use of laser induced post-ionization (MALDI-2) combined with trapped ion mobility separation (TIMS) and orthogonal time-of-flight (QTOF) MS for the visualization of isobaric derivatized androgens in murine tumour xenograft at about 50 µm spatial resolution. With this combination, isobaric T and DHEA were separated in tissue sections and the signals of derivatized steroids enhanced by about 20 times. The combination of TIMS and MALDI-2 thus shows unique potential to study tissue intracrinology within target tissues. This could offer the opportunity for many novel insights into tissue-specific androgen biology.

Molecular insights into symbiosis-mapping sterols in a marine flatworm-algae-system using high spatial resolution MALDI-2-MS imaging with ion mobility separation.

Waminoa sp. acoel flatworms hosting Symbiodiniaceae and the related Amphidinium dinoflagellate algae are an interesting model system for symbiosis in marine environments. While the host provides a microhabitat and safety, the algae power the system by photosynthesis and supply the worm with nutrients. Among these nutrients are sterols, including cholesterol and numerous phytosterols. While it is widely accepted that these compounds are produced by the symbiotic dinoflagellates, their transfer to and fate within the sterol-auxotrophic Waminoa worm host as well as their role in its metabolism are unknown. Here we used matrix-assisted laser desorption ionization (MALDI) mass spectrometry imaging combined with laser-induced post-ionization and trapped ion mobility spectrometry (MALDI-2-TIMS-MSI) to map the spatial distribution of over 30 different sterol species in sections of the symbiotic system. The use of laser post-ionization crucially increased ion yields and allowed the recording of images with a pixel size of 5 µm. Trapped ion mobility spectrometry (TIMS) helped with the tentative assignment of over 30 sterol species. Correlation with anatomical features of the worm, revealed by host-derived phospholipid signals, and the location of the dinoflagellates, revealed by chlorophyll a signal, disclosed peculiar differences in the distribution of different sterol species (e.g. of cholesterol versus stigmasterol) within the receiving host. These findings point to sterol species-specific roles in the metabolism of Waminoa beyond a mere source of energy. They also underline the value of the MALDI-2-TIMS-MSI method to future research in the spatially resolved analysis of sterols.

MALDI-2 for the Enhanced Analysis of N-Linked Glycans by Mass Spectrometry Imaging.

N-glycans are important players in a variety of pathologies including different types of cancer, (auto)immune diseases, and also viral infections. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is an important tool for high-throughput N-glycan profiling and, upon use of tandem MS, for structure determination. By use of MALDI-MS imaging (MSI) in combination with PNGase F treatment, also spatially correlated N-glycan profiling from tissue sections becomes possible. Here we coupled laser-induced postionization, or MALDI-2, to a trapped ion mobility quadrupole time-of-flight mass spectrometer (timsTOF fleX MALDI-2, Bruker Daltonics). We demonstrate that with MALDI-2 the sensitivity for the detection of molecular [M - H]- species of N-glycans increased by about 3 orders of magnitude. Compared to the current gold standard, the positive ion mode analysis of [M + Na]+ adducts, a sensitivity increase by about a factor of 10 is achieved. By exploiting the advantageous fragmentation behavior of [M - H]- ions, exceedingly rich structural information on the composition of complex N-glycans was moreover obtained directly from thin tissue sections of human cerebellum and upon use of low-energy collision-induced dissociation tandem MS. In another set of experiments, in this case by use of a modified Synapt G2-S QTOF mass spectrometer (Waters), we investigated the influence of relevant input parameters, in particular pressure of the N2 cooling gas in the ion source, delay between the two laser pulses, and that of their pulse energies. In this way, analytical conditions were identified at which molecular ion abundances were maximized and fragmentation reactions minimized. The use of negative ion mode MALDI-2-MSI could constitute a valuable tool in glycobiology research.

Low-Pressure Photoionization in a Dual-Ion Funnel Injector Coupled to an Orbitrap Mass Spectrometer for Direct Analysis of Human Breath and Head-Space Sampled Coffee Roasts.

Low-pressure photoionization (LPPI) is a versatile tool for the mass spectrometric detection of (semi-)volatile organic compounds, (s)VOC. Here, a dual-ion funnel MALDI/ESI ion injector was equipped with a direct-inlet LPPI module. A radio-frequency (RF) drive enabled the implementation of three Kr discharge lamps in a novel design optimized for efficient photoionization and undisturbed ion trajectories. Supported by expansion and collisional cooling and, optionally, dopant vapor, primarily intact radical ions and protonated molecules were generated. Molecular identification was supported by the high-resolving power of an Orbitrap mass analyzer. In our proof-of-concept study, exhaled human breath and head-space sampled coffee grounds were characterized with this high-throughput technique. From breath, a few hundred and for the coffee roasts more than thousand distinct (s)VOC features were recorded. Principal component analysis enabled the differentiation of coffee grounds by origin and roasting protocol.

Detailed Characterization of the Postionization Efficiencies in MALDI-2 as a Function of Relevant Input Parameters.

A recently introduced technique based on MALDI with laser-induced postionization (PI), also named MALDI-2, increases the ion yields for numerous classes of lipids, metabolites, and carbohydrates in MALDI-MS imaging experiments under certain experimental conditions. Here, we used a semiautomatic LabVIEW-based protocol to investigate and optimize the efficiency of the PI process dependent on four relevant input parameters and a dense parameter grid: pulse energies of the two lasers, delay between the laser pulses, and buffer gas pressure in the ion source. All experiments were conducted with a modified MALDI-2 Synapt G2-S mass spectrometer (Waters) and use of a focal spot size on the sample of 15-17 µm. A wavelength-tunable optical parametric oscillator (OPO) laser served for PI at 260 or 280 nm. The investigated MALDI matrices were: 2,5-dihydroxybenzoic acid (positive ion mode, +), 2,5-dihydroxyacetophenone (+), α-cyano-4-hydroxycinnamic acid (+), norharmane (negative-ion mode, -), and 1,5-diaminonapthalene (-). A porcine brain extract served as lipid standard. In the positive-ion mode, a maximum boost for the generated [M + H]+ species was found with a N2 buffer gas pressure of ∼2 mbar and a delay between the laser emissions of ∼10 µs. Higher optimal delay settings of several 10 µs were registered for the two studied matrices in negative-ion mode. With regard to the laser fluences, best PI efficiencies were reached using maximum available ablation and PI laser pulse energies of up to 25 and 160 µJ, respectively. For analytes not profiting from MALDI-2, best ion signal yields were recorded for ablation laser pulse energies of around 7 µJ, depending on the MALDI matrix. At higher laser pulse energies, sizable fragmentation is observed for these ions. The PI laser pulse energy did not have any influence on the ion signals of these species. For optimal ion yield of all analyte species, best results were obtained with an ablation laser pulse energy of ∼7 µJ and a PI laser pulse energy of ∼160 µJ. Our comprehensive data set provides valuable insight into the mechanisms underlying the MALDI-2 processes and could help to further optimize this emerging technique.

MALDI-2 on a Trapped Ion Mobility Quadrupole Time-of-Flight Instrument for Rapid Mass Spectrometry Imaging and Ion Mobility Separation of Complex Lipid Profiles.

Matrix-assisted laser desorption/ionization combined with laser-induced postionization (MALDI-2) is a recently introduced method for enhanced mass spectrometry imaging of numerous classes of biomolecules, including phospho- and glycolipids in tissue sections at high lateral resolution. Here we describe the first adaptation of the technology to a Bruker timsTOF fleX mass spectrometer. Upon use of a 1 kHz postionization laser, MALDI-2 produces a sizable increase in the number of detected features as well as in ion signal intensities. This enhancement is similar to that described previously for low repetition rate MALDI-2 systems, but now enables substantially enhanced measurement speeds. In our proof-of-concept study, we furthermore demonstrate, on examples of rat brain and testis tissue sections, that the combination of MALDI-2 with the trapped ion mobility spectrometry (TIMS) functionality of the instrument can crucially support unravelling the complex molecular composition of the lipidome. Numerous isomeric/isobaric ion species are successfully separated upon using the collisional cross section (CCS) as additional specific physical property. With the possibilities of high data acquisition speed or high separation powers in combination with the increased sensitivity of MALDI-2 available in one instrument, the described methodology could be a valuable tool in many areas of biological and medical research.

Ozonization of Tissue Sections for MALDI MS Imaging of Carbon-Carbon Double Bond Positional Isomers of Phospholipids.

Visualizing the differential distribution of carbon-carbon double bond (C═C db) positional isomers of unsaturated phospholipids (PL) in tissue sections by use of refined matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI MSI) technologies offers a high promise to deeper understand PL metabolism and isomer-specific functions in health and disease. Here we introduce an on-tissue ozonization protocol that enables a particular straightforward derivatization of unsaturated lipids in tissue sections. Collision-induced dissociation (CID) of MALDI-generated ozonide ions (with yields in the several ten percent range) produced the Criegee fragment ion pairs, which are indicative of C═C db position(s). We used our technique for visualizing the differential distribution of Δ9 and Δ11 isomers of phosphatidylcholines in mouse brain and in human colon samples with the desorption laser spot size 15 µm, emphasizing the potential of the technique to expose local isomer-specific metabolism of PLs.

MALDI-2 Mass Spectrometry and Immunohistochemistry Imaging of Gb3Cer, Gb4Cer, and Further Glycosphingolipids in Human Colorectal Cancer Tissue.

The main cellular receptors of Shiga toxins (Stxs), the neutral glycosphingolipids (GSLs), globotriaosylceramide (Gb3Cer/CD77) and globotetraosylceramide (Gb4Cer), are significantly upregulated in about half of the human colorectal carcinomas (CRC) and in other cancers. Therefore, conjugates exploiting the Gb3Cer/Gb4Cer-binding B subunit of Stx (StxB) have attracted great interest for both diagnostic and adjuvant therapeutic interventions. Moreover, fucosylated GSLs were recognized as potential tumor-associated targets. One obstacle to a broader use of these receptor/ligand systems is that the contribution of specific GSLs to tumorigenesis, in particular, in the context of an altered lipid metabolism, is only poorly understood. A second is that also nondiseased organs (e.g., kidney) and blood vessels can express high levels of certain GSLs, not least Gb3Cer/Gb4Cer. Here, we used, in a proof-of-concept study, matrix-assisted laser desorption/ionization mass spectrometry imaging combined with laser-induced postionization (MALDI-2-MSI) to simultaneously visualize the distribution of several Gb3Cer/Gb4Cer lipoforms and those of related GSLs (e.g., Gb3Cer/Gb4Cer precursors and fucosylated GSLs) in tissue biopsies from three CRC patients. Using MALDI-2 and StxB-based immunofluorescence microscopy, Gb3Cer and Gb4Cer were mainly found in dedifferentiated tumor cell areas, tumor stroma, and tumor-infiltrating blood vessels. Notably, fucosylated GSL such as Fuc-(n)Lc4Cer generally showed a highly localized expression in dysplastic glands and indian file-like cells infiltrating adipose tissue. Our "molecular histology" approach could support stratifying patients for intratumoral GSL expression to identify an optimal therapeutic strategy. The improved chemical coverage by MALDI-2 can also help to improve our understanding of the molecular basis of tumor development and GSL metabolism.