Assessment of MALDI matrices for the detection and visualization of phosphatidylinositols and phosphoinositides in mouse kidneys through matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI).

Phosphatidylinositols and their phosphorylated derivatives, known as phosphoinositides, are crucial in cellular processes, with their abnormalities linked to various diseases. Thus, identifying and measuring phosphoinositide levels in tissues are crucial for understanding their contributions to cellular processes and disease development. One powerful technique for mapping the spatial distribution of molecules in biological samples is matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI). This technique allows for the simultaneous detection and analysis of multiple lipid classes in situ, making it invaluable for unbiased lipidomic studies. However, detecting phosphoinositides with MALDI-MSI is challenging due to their relatively low abundance in tissues and complex matrix effects. Addressing this, our study focused on optimizing matrix selection and thickness for better detection of phosphatidylinositols and their phosphorylated forms in mouse kidney tissues. Various matrices were assessed, including 9AA, DAN, CMBT, and DHA, adjusting their coating to improve ionization efficiency. Our results demonstrate that DAN, DHA, and CMBT matrices produced high-intensity chemical images of phosphatidylinositol distributions within kidney sections. These matrices, particularly DAN, DHA, and CMBT, allowed the identification of even low-abundance phosphoinositides, through tentative identifications. Notably, DAN and DHA served as optimal candidates due to their prominent detection and ability to map a majority of phosphatidylinositol species, while CMBT showed potential detection capability for phosphatidylinositol triphosphate compounds. These findings not only provide valuable insights for future research on the involvement of phosphoinositides in kidney pathophysiology, but also propose the use of the identified optimal matrices, particularly DAN and DHA, as the preferred choices for enhanced detection and mapping of these lipid species in future studies.

Sublimation application of 5-chloro-2-mercaptobenzothiazole matrix for matrix-assisted laser desorption/ionization mass spectrometry imaging of mouse kidney.

RATIONALE: Sublimation is a solvent-free technique used to apply a uniform matrix coating over a large sample plate, improving the matrix's purity and enhancing the analyte signal. Although the 5-chloro-2-mercaptobenzothiazole (CMBT) matrix was introduced years ago, there are no reports of its application via sublimation. We investigated the experimental parameters that are optimal for CMBT matrix sublimation on mouse kidney samples. We also evaluated the stability of the sublimed CMBT matrix under a vacuum environment. Using kidney samples prepared with a sublimated CMBT matrix, we conducted matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) analysis of specific phospholipids (phosphatidylcholine and phosphatidylglycerol in the positive ion mode and phosphatidylinositol in the negative ion mode). We also explored various spatial resolutions (50, 20, and 10 µm) and performed sequential MALDI-hematoxylin and eosin (H&E) staining. METHODS: The CMBT matrix was applied to kidney samples using a sublimation apparatus connected to a vacuum pump to achieve a pressure of 0.05 Torr. The matrix was then subjected to different temperatures and sublimation times to determine the optimal conditions for matrix application. Subsequently, a Q-Exactive mass spectrometer equipped with a Spectroglyph MALDI ion source was employed for MALDI-MSI experiments. Standard protocols were followed for H&E staining after MALDI analysis. RESULTS: A matrix thickness of 0.15 mg/cm2 yielded high-quality images. The sublimated matrix exhibited minimal loss after approximately 20 h of exposure to a vacuum of 7 Torr, indicating its stability under these conditions. Ion images were successfully obtained at spatial resolutions of 50, 20, and 10 µm. Furthermore, orthogonal histological information was obtained through sequential MALDI-H&E staining. CONCLUSIONS: We demonstrate that samples prepared for MALDI-MSI using sublimation to apply the CMBT matrix yield high-quality mass spectrometric images of mouse kidney sections. We also provide data for the impact of various experimental parameters on image quality (e.g., temperature, time, matrix thickness, and spatial resolution).

Internal standard application strategies in mass spectrometry imaging by desorption electrospray ionization mass spectrometry.

RATIONALE: We developed a model case study to evaluate three internal standard (IS) application strategies (methods I-III) using the psycholeptic phenobarbital (PB) and the isotopically labelled IS phenobarbital-D5 (PB-D5) from in vitro dosed tissues of the golden apple snail (Pomacea diffusa) by desorption electrospray ionization mass spectrometry imaging (DESI-MSI). METHODS: In method I, the IS was deposited as microspots on top of 10 µm thick snail tissues; in method II, a thin IS film was applied; and in method III, the IS was spiked into the DESI solvent spray. DESI-MSI analyses were performed using a Thermo LTQ mass spectrometer equipped with a custom-built DESI source and two-dimensional moving stage. PB (m/z 231) and PB-D5 (m/z 236) were monitored in selected ion monitoring mode between m/z 227 and 239. RESULTS: The analytical performance of two IS strategies (methods I and II) in DESI-MSI was evaluated based on an intra- and inter-day precision assay, an accuracy assessment, and statistical analysis. In the inter-day DESI-MSI assay, method I exhibited better precision (6.5%-7.4%) than method II (10.7%-17.6%) between 10 and 100 ng/µL. In the accuracy assessment, PB quality controls of 75 ng/µL were back-calculated as 71 ± 4 and 83 ± 9 ng/µL, resulting in relative errors of -5% and 11% for methods I and II, respectively. Method III did not work under the experimental design and was not evaluated. CONCLUSIONS: Three IS application strategies were investigated and compared for a routine quantitative DESI-MSI approach. Methods I and II were not statistically significantly different as shown by a Bland-Altman plot, suggesting that these two methods can be used interchangeably. However, method III requires further research for future quantitative DESI-MSI analyses.

Calycopterin, a major flavonoid from Marcetia latifolia, modulates virulence-related traits in Pseudomonas aeruginosa.

Although bacterial resistance is a worldwide growing concern, the development of bacteriostatic and bactericidal drugs has been decreasing in the last decade. Compounds that modulate the microorganism virulence, without killing it, have been considered promising alternatives to combat bacterial infections. However, most signaling pathways that regulate virulence are complex and not completely understood. The rich chemical diversity of natural products offers a good starting point to identify key compounds that shed some light on this matter. Therefore, we investigated the role of Marcetia latifolia ethanolic extract, as well as its major constituent, calycopterin (5,4'-dihydroxy-3,6,7,8-tetramethoxylflavone), in the regulation of virulence-related phenotypes of Pseudomonas aeruginosa. Our results show that calycopterin inhibits pyocyanin production (EC50 = 32 µM), reduces motility and increases biofilm formation in a dose-dependent manner. Such biological profile suggests that calycopterin modulates targets that may act upstream the quorum sensing regulators and points to its utility as a chemical probe to further investigate P. aeruginosa transition from planktonic to sessile lifestyle.

Fragmentation study of clerodane diterpenes from Casearia species by tandem mass spectrometry (quadrupole time-of-flight and ion trap).

RATIONALE: Clerodane-type diterpenes from Casearia species show important pharmacological activites such as antitumor, antimicrobial and anti-inflamatory. There are several mass spectrometry (MS)-based methods for identification of diterpenes; however, there is still a lack of MS procedures capable of providing characteristic fragmentation pathways for a rapid and unambiguous elucidation of casearin-like compounds. METHODS: Casearin-like compounds were investigated by electrospray ionization tandem mass spectrometry (ESI-MS/MS). The fragmentation studies were carried out by tandem mass spectrometry in space (quadrupole time-of-flight (QTOF)) using different collision energies and also by tandem mass spectrometry in time (QIT) by selective isolation of product ions. RESULTS: Casearin-like compounds presented a predominance of sodium- and potassium-cationized precursor ions. Both QIT and QTOF techniques provided sequential neutral losses of esters related to the R1 to R5 substituents linked to the nucleus of the clerodane diterpenes. The fragmentation pathway is initiated with a cleavage of the ester moieties R2 followed by the elimination of the ester groups R3 , both losing neutral carboxylic acids. Using QIT, it was also possible to observe the cleavage of the ester groups R1 or R5 by MS4 experiments. CONCLUSIONS: Through a rational analysis of the fragmentation mechanisms of Casearia diterpenes it was possible to suggest an annotation strategy based on the sequential cleavages of the ester groups related to the R2 , R3 and R5 substituents. These results will assist studies of the dereplication and metabolomics involving casearin-like compounds present in complex extracts of Casearia species.

Rapid structural characterisation of benzylisoquinoline and aporphine alkaloids from Ocotea spixiana acaricide extract by HPTLC-DESI-MSn.

INTRODUCTION: Lauraceae alkaloids are a structurally diverse class of plant specialised secondary metabolites that play an important role in modern pharmacotherapy, being useful as well as model compounds for the development of synthetic analogues. However, alkaloids characterisation is challenging due to low concentrations, the complexity of plant extracts, and long processes for accurate structural determinations. OBJECTIVE: The use of high-performance thin layer chromatography coupled with desorption electrospray ionisation multistage mass spectrometry (HPTLC DESI-MSn ) as a fast tool to identify alkaloids present in Ocotea spixiana extract and evaluate the extract's acaricide activity. METHODS: Ocotea spixiana twigs were extracted by conventional liquid-liquid partitioning. HPTLC analysis of the ethyl acetate extract was performed to separate isobaric alkaloids prior to DESI-MSn analysis, performed from MS3 up to MS7 . The extract's acaricide activity against Rhipicephalus microplus was evaluated by in vitro (larval immersion test) and in silico tests. RESULTS: HPTLC-DESI-MSn analysis was performed to identify a total of 13 aporphine and four benzylisoquinoline-type alkaloids reported for the first time in O. spixiana. In vitro evaluation of the extract and the alkaloid boldine showed significant activity against R. microplus larvae. It was established in silico that boldine had important intermolecular interactions with R. microplus acetylcholinesterase enzyme. CONCLUSION: The present study demonstrated that HPTLC-DESI-MSn is a useful analytical tool to identify isoquinoline alkaloids in plant extracts. The acaricide activity of the O. spixiana ethyl acetate extract can be correlated to the presence of alkaloids.

Review and perspectives on the applications of mass spectrometry imaging under ambient conditions.

Ambient mass spectrometry (AMS)-based techniques are performed under ambient conditions in which the ionization and desorption occur in the open environment allowing the direct analysis of molecules with minimal or no sample preparation. A selected group of AMS techniques demonstrate imaging capabilities that can provide information about the localization of molecules on complex sample surfaces such as biological tissues. 2D, 3D, and multimodal imaging have unlocked an array of applications to systematically address complex problems in many areas of research such as drug monitoring, natural products, forensics, and cancer diagnostics. In the present review, we summarize recent advances in the field with respect to the implementation of new ambient ionization techniques and current applications in the last 5 years. In more detail, we mainly focus on imaging applications in topics related to animal whole bodies and tissues, single cells, cancer diagnostics and biomarkers, microbial cultures and co-cultures, plant and natural product metabolomics, and forensic applications. Finally, we discuss new areas of research, future perspectives, and the overall direction that the field may take in the years to come.

Characterization and mapping of secondary metabolites of Streptomyces sp. from caatinga by desorption electrospray ionization mass spectrometry (DESI-MS).

The discovery of new secondary metabolites is a challenge to biotechnologists due to the emergence of superbugs and drug resistance. Knowledge about biodiversity and the discovery of new microorganisms have become major objectives; thus, new habitats like extreme ecosystems have become places of interest to research. In this context, caatinga is an unexplored biome. The ecosystem caatinga is a rich habitat for thermophilic microbes. Its high temperature and dry climate cause selective microbes to flourish and become established. Actinobacteria (Caat 1-54 genus Streptomyces sp.) isolated from the soil of caatinga was investigated to characterize and map its secondary metabolites by desorption electrospray ionization mass spectrometry imaging (DESI-MSI). With this technique, the production of bioactive metabolites was detected and associated with the different morphological differentiation stages within a typical Streptomyces sp. life cycle. High-resolution mass spectrometry, tandem mass spectrometry, UV-Vis profiling and NMR analysis were also performed to characterize the metabolite ions detected by DESI-MS. A novel compound, which is presumed to be an analogue of the antifungal agent lienomycin, along with the antimicrobial compound lysolipin I were identified in this study to be produced by the bacterium. The potency of these bioactive compounds was further studied by disc diffusion assays and their minimum inhibitory concentrations (MIC) against Bacillus and Penicillium were determined. These bioactive metabolites could be useful to the pharmaceutical industry as candidate compounds, especially given growing concern about increasing resistance to available drugs with the emergence of superbugs. Consequently, the unexplored habitat caatinga affords new possibilities for novel bioactive compound discovery. Graphical Abstract ᅟ.

Detection and imaging of thermochromic ink compounds in erasable pens using desorption electrospray ionization mass spectrometry.

RATIONALE: Thermochromic ink pens are widely accessible worldwide and have gained popularity among the general public. These pens are very useful to undo mistakes while writing important documents or exams. They are also, however, misused in committing crimes such as counterfeiting checks or wills. Thus, the forensics community is in need of techniques that will allow these forgeries to be detected rapidly, reliably and conveniently. METHODS: Thermochromic ink compounds were investigated using Desorption Electrospray Ionization (DESI) coupled with an LTQ mass spectrometer and Thin-Layer Chromatography (TLC). Tandem mass spectrometric analysis was conducted using Electrospray Ionization (ESI) coupled with an Orbitrap LTQ mass spectrometer performing Collision-Induced Dissociation (CID) for identification of ink traces. RESULTS: Chemical marker ions characteristic of the state of ink (visible or invisible) were identified and mapped in ink traces by the use of DESI-MS imaging. These ions can be employed by forensic experts as fingerprint markers in forged documents. The marker ions were also characterised by conducting tandem mass spectrometry using paper spray in an Orbitrap LTQ mass spectrometer. CONCLUSIONS: Specific chemical components yielding ions of m/z 400, 405, 615 and 786 were distinguished as only being apparent in the invisible and reappeared state of the ink. The absence of these compounds in the original state of the ink enabled their recognition as useful chemical determinants in detecting forgery. DESI-MSI was thus shown to be a very useful, convenient and reliable technique for detecting forgery in paper documents due to its fast and reproducible mode of analysis, with no sample preparation and minimal damage to the document under investigation. Copyright © 2017 John Wiley & Sons, Ltd.

Correction: Rapid determination of the tumour stroma ratio in squamous cell carcinomas with desorption electrospray ionization mass spectrometry (DESI-MS): a proof-of-concept demonstration.

Correction for 'Rapid determination of the tumour stroma ratio in squamous cell carcinomas with desorption electrospray ionization mass spectrometry (DESI-MS): a proof-of-concept demonstration' by Michael Woolman et al., Analyst, 2017, 142, 3250-3260.

Rapid determination of the tumour stroma ratio in squamous cell carcinomas with desorption electrospray ionization mass spectrometry (DESI-MS): a proof-of-concept demonstration.

Squamous cell carcinomas constitute a major class of head & neck cancers, where the tumour stroma ratio (TSR) carries prognostic information. Patients affected by stroma-rich tumours exhibit a poor prognosis and a higher chance of relapse. As such, there is a need for a technology platform that allows rapid determination of the tumour stroma ratio. In this work, we provide a proof-of-principle demonstration that Desorption Electrospray Ionization Mass Spectrometry (DESI-MS) can be used to determine tumour stroma ratios. Slices from three independent mouse xenograft tumours from the human FaDu cell line were subjected to DESI-MS imaging, staining and detailed analysis using digital pathology methods. Using multivariate statistical methods we compared the MS profiles with those of isolated stromal cells. We found that m/z 773.53 [PG(18:1)(18:1) - H]-, m/z 835.53 [PI(34:1) - H]- and m/z 863.56 [PI(18:1)(18:0) - H]- are biomarker ions that can distinguish FaDu cancer from cancer associated fibroblast (CAF) cells. A comparison with DESI-MS analysis of controlled mixtures of the CAF and FaDu cells showed that the abundance of the biomarker ions above can be used to determine, with an error margin of close to 5% compared with quantitative pathology estimates, TSR values. This proof-of-principle demonstration is encouraging and must be further validated using human samples and a larger sample base. At maturity, DESI-MS thus may become a stand-alone molecular pathology tool providing an alternative rapid cancer assessment without the need for time-consuming staining and microscopy methods, potentially further conserving human resources.

Ambient Ionization Mass Spectrometry for Cancer Diagnosis and Surgical Margin Evaluation.

BACKGROUND: There is a clinical need for new technologies that would enable rapid disease diagnosis based on diagnostic molecular signatures. Ambient ionization mass spectrometry has revolutionized the means by which molecular information can be obtained from tissue samples in real time and with minimal sample pretreatment. New developments in ambient ionization techniques applied to clinical research suggest that ambient ionization mass spectrometry will soon become a routine medical tool for tissue diagnosis. CONTENT: This review summarizes the main developments in ambient ionization techniques applied to tissue analysis, with focus on desorption electrospray ionization mass spectrometry, probe electrospray ionization, touch spray, and rapid evaporative ionization mass spectrometry. We describe their applications to human cancer research and surgical margin evaluation, highlighting integrated approaches tested for ex vivo and in vivo human cancer tissue analysis. We also discuss the challenges for clinical implementation of these tools and offer perspectives on the future of the field. SUMMARY: A variety of studies have showcased the value of ambient ionization mass spectrometry for rapid and accurate cancer diagnosis. Small molecules have been identified as potential diagnostic biomarkers, including metabolites, fatty acids, and glycerophospholipids. Statistical analysis allows tissue discrimination with high accuracy rates (>95%) being common. This young field has challenges to overcome before it is ready to be broadly accepted as a medical tool for cancer diagnosis. Growing research in new, integrated ambient ionization mass spectrometry technologies and the ongoing improvements in the existing tools make this field very promising for future translation into the clinic.

Reactive DESI-MS imaging of biological tissues with dicationic ion-pairing compounds.

This work illustrates reactive desorption electrospray ionization mass spectrometry (DESI-MS) with a stable dication on biological tissues. Rat brain and zebra fish tissues were investigated with reactive DESI-MS in which the dictation forms a stable bond with biological tissue fatty acids and lipids. Tandem mass spectrometry (MS/MS) was used to characterize the dication (DC9) and to identify linked lipid-dication compounds formed. The fragment m/z 85 common to both DC9 fragmentation and DC9-lipid fragmentation was used to confirm that DC9 is indeed bonded with the lipids. Lipid signals in the range of m/z 250-350 and phosphoethanolamines (PE) m/z 700-800 observed in negative ion mode were also detected in positive ion mode with reactive DESI-MS with enhanced signal intensity. Reactive DESI-MS imaging in positive ion mode of rat brain and zebra fish tissues allowed enhanced detection of compounds commonly observed in the negative ion mode.

Contrast Agent Mass Spectrometry Imaging Reveals Tumor Heterogeneity.

Mapping intratumoral heterogeneity such as vasculature and margins is important during intraoperative applications. Desorption electrospray ionization mass spectrometry (DESI-MS) has demonstrated potential for intraoperative tumor imaging using validated MS profiles. The clinical translation of DESI-MS into a universal label-free imaging technique thus requires access to MS profiles characteristic to tumors and healthy tissues. Here, we developed contrast agent mass spectrometry imaging (CA-MSI) that utilizes a magnetic resonance imaging (MRI) contrast agent targeted to disease sites, as a label, to reveal tumor heterogeneity in the absence of known MS profiles. Human breast cancer tumors grown in mice were subjected to CA-MSI using Gadoteridol revealing tumor margins and vasculature from the localization of [Gadoteridol+K](+) and [Gadoteridol+Na](+) adducts, respectively. The localization of the [Gadoteridol+K](+) adduct as revealed through DESI-MS complements the in vivo MRI results. DESI-MS imaging is therefore possible for tumors for which no characteristic MS profiles are established. Further DESI-MS imaging of the flux of the contrast agent through mouse kidneys was performed indicating secretion of the intact label.

Imprint Desorption Electrospray Ionization Mass Spectrometry Imaging for Monitoring Secondary Metabolites Production during Antagonistic Interaction of Fungi.

Direct analysis of microbial cocultures grown on agar media by desorption electrospray ionization mass spectrometry (DESI-MS) is quite challenging. Due to the high gas pressure upon impact with the surface, the desorption mechanism does not allow direct imaging of soft or irregular surfaces. The divots in the agar, created by the high-pressure gas and spray, dramatically change the geometry of the system decreasing the intensity of the signal. In order to overcome this limitation, an imprinting step, in which the chemicals are initially transferred to flat hard surfaces, was coupled to DESI-MS and applied for the first time to fungal cocultures. Note that fungal cocultures are often disadvantageous in direct imaging mass spectrometry. Agar plates of fungi present a complex topography due to the simultaneous presence of dynamic mycelia and spores. One of the most devastating diseases of cocoa trees is caused by fungal phytopathogen Moniliophthora roreri. Strategies for pest management include the application of endophytic fungi, such as Trichoderma harzianum, that act as biocontrol agents by antagonizing M. roreri. However, the complex chemical communication underlying the basis for this phytopathogen-dependent biocontrol is still unknown. In this study, we investigated the metabolic exchange that takes place during the antagonistic interaction between M. roreri and T. harzianum. Using imprint-DESI-MS imaging we annotated the secondary metabolites released when T. harzianum and M. roreri were cultured in isolation and compared these to those produced after 3 weeks of coculture. We identified and localized four phytopathogen-dependent secondary metabolites, including T39 butenolide, harzianolide, and sorbicillinol. In order to verify the reliability of the imprint-DESI-MS imaging data and evaluate the capability of tape imprints to extract fungal metabolites while maintaining their localization, six representative plugs along the entire M. roreri/T. harzianum coculture plate were removed, weighed, extracted, and analyzed by liquid chromatography-high-resolution mass spectrometry (LC-HRMS). Our results not only provide a better understanding of M. roreri-dependent metabolic induction in T. harzianum, but may seed novel directions for the advancement of phytopathogen-dependent biocontrol, including the generation of optimized Trichoderma strains against M. roreri, new biopesticides, and biofertilizers.

Ambient Mass Spectrometry Imaging with Picosecond Infrared Laser Ablation Electrospray Ionization (PIR-LAESI).

A picosecond infrared laser (PIRL) is capable of cutting through biological tissues in the absence of significant thermal damage. As such, PIRL is a standalone surgical scalpel with the added bonus of minimal postoperative scar tissue formation. In this work, a tandem of PIRL ablation with electrospray ionization (PIR-LAESI) mass spectrometry is demonstrated and characterized for tissue molecular imaging, with a limit of detection in the range of 100 nM for reserpine or better than 5 nM for verapamil in aqueous solution. We characterized PIRL crater size using agar films containing Rhodamine. PIR-LAESI offers a 20-30 µm vertical resolution (∼3 µm removal per pulse) and a lateral resolution of ∼100 µm. We were able to detect 25 fmol of Rhodamine in agar ablation experiments. PIR-LAESI was used to map the distribution of endogenous methoxykaempferol glucoronide in zebra plant (Aphelandra squarrosa) leaves producing a localization map that is corroborated by the literature. PIR-LAESI was further used to image the distribution inside mouse kidneys of gadoteridol, an exogenous magnetic resonance contrast agent intravenously injected. Parallel mass spectrometry imaging (MSI) using desorption electrospray ionization (DESI) and matrix assisted laser desorption ionization (MALDI) were performed to corroborate PIR-LAESI images of the exogenous agent. We further show that PIR-LAESI is capable of desorption ionization of proteins as well as phospholipids. This comparative study illustrates that PIR-LAESI is an ion source for ambient mass spectrometry applications. As such, a future PIRL scalpel combined with secondary ionization such as ESI and mass spectrometry has the potential to provide molecular feedback to guide PIRL surgery.

High-performance thin-layer chromatography/desorption electrospray ionization mass spectrometry imaging of the crude extract from the peels of Citrus aurantium L. (Rutaceae).

RATIONALE: Citrus aurantium L. is a plant belonging to the Rutaceae family, whose extracts are extensively used in weight management products and as thermogenic agents. Here we present two methodologies to analyse the extracts obtained from the peels of Citrus aurantium L. that usually require multiple sample preparation and detection steps. METHODS: Polar compounds of the crude extract from the peels of Citrus aurantium L. (Rutaceae) were investigated by direct infusion electrospray ionization mass spectrometry (ESI-MS) and high-performance thin-layer chromatography (HPTLC) coupled to desorption electrospray ionization mass spectrometry (DESI-MS). ESI-MS was performed in both positive and negative ion modes. Molecular imaging of the HPTLC plates was used for the direct analysis of the phytocompounds present in the crude extract from the peels of Citrus aurantium L. by DESI-MS imaging. RESULTS: Characteristic mass spectra with many diagnostic ions were obtained from the extract analysis, allowing a fast and reliable identification of these species. Tandem mass spectrometry (MS/MS) was employed to confirm the identity of specific metabolites. CONCLUSIONS: HPTLC/DESI-MS imaging is a relatively fast, versatile, and efficient technique for natural product analysis, since many more ions are observed than with the direct infusion ESI-MS. The MS/MS technique provided information about the component structures, revealing the presence of important bioactive components. The application of DESI-MS imaging may contribute to the improvement identification and characterization of pharmacologically active compounds in phytochemistry.

Functionalized porous silicon surfaces as DESI-MS substrates for small molecules analysis.

In desorption electrospray ionization mass spectrometry (DESI-MS), the type of surface in addition to low gas and solvent flow rates help to avoid the "splashing of solvent" or "washing effect", by which samples are promptly removed from the surface by the spray. These effects operate on smooth surfaces and generally result in unstable signals as the spray moves over the spot. The aim of this work is to compare the performance of functionalized porous silicon surfaces (pSi) for small molecules analysis with regard to the stability of the signal and the limits of detection (LODs) observed in DESI-MS. The results showed that functional groups, like 1-decene and heptadecafluoro-1,1,2,2-tetrahydrodecyl trimethoxysilane, on pSi surface provides a good alternative for dried spot analysis by DESI-MS, improving stability of the signal and the LODs. This improvement is possible because the dual process containing the weak sample-surface interactions of the hydrophobic characteristic of the functional groups and increasing the surface area of interaction between the sample and the thin solvent film created by the DESI spray, resulting in more effective dissolution of the analyte in the spray solvent without fast removal of the sample.

Mass spectrometry imaging under ambient conditions.

Mass spectrometry imaging (MSI) has emerged as an important tool in the last decade and it is beginning to show potential to provide new information in many fields owing to its unique ability to acquire molecularly specific images and to provide multiplexed information, without the need for labeling or staining. In MSI, the chemical identity of molecules present on a surface is investigated as a function of spatial distribution. In addition to now standard methods involving MSI in vacuum, recently developed ambient ionization techniques allow MSI to be performed under atmospheric pressure on untreated samples outside the mass spectrometer. Here we review recent developments and applications of MSI emphasizing the ambient ionization techniques of desorption electrospray ionization (DESI), laser ablation electrospray ionization (LAESI), probe electrospray ionization (PESI), desorption atmospheric pressure photoionization (DAPPI), femtosecond laser desorption ionization (fs-LDI), laser electrospray mass spectrometry (LEMS), infrared laser ablation metastable-induced chemical ionization (IR-LAMICI), liquid microjunction surface sampling probe mass spectrometry (LMJ-SSP MS), nanospray desorption electrospray ionization (nano-DESI), and plasma sources such as the low temperature plasma (LTP) probe and laser ablation coupled to flowing atmospheric-pressure afterglow (LA-FAPA). Included are discussions of some of the features of ambient MSI for example the ability to implement chemical reactions with the goal of providing high abundance ions characteristic of specific compounds of interest and the use of tandem mass spectrometry to either map the distribution of targeted molecules with high specificity or to provide additional MS information on the structural identification of compounds. We also describe the role of bioinformatics in acquiring and interpreting the chemical and spatial information obtained through MSI, especially in biological applications for tissue diagnostic purposes. Finally, we discuss the challenges in ambient MSI and include perspectives on the future of the field.

Imaging of whole zebra fish (Danio rerio) by desorption electrospray ionization mass spectrometry.

RATIONALE: To demonstrate the potential use of zebra fish (Danio rerio) as a model vertebrate organism by producing two-dimensional ion images of the whole zebra fish, and being able to distinguish particular areas of interest such as the brain, spinal cord, and stomach region using a desorption electrospray ionization (DESI) ion source coupled to a linear ion trap. METHODS: Imaging experiments are performed on 45 µm sagittal slices of zebra fish (Danio rerio), which are thaw-mounted onto microscope glass slides. The slides are then analyzed using a solvent of acetonitrile/dimethylformamide (50:50) (ACN/DMF), with a solvent flow rate of 1.5 µL/min; data are acquired in negative ion mode. Raw mass spectrum data files are converted into a readable file for Biomap. The images produced are then analyzed for ion distributions. RESULTS: We are able to create clear, distinct, chemical intensity images of the brain, spinal cord, and stomach based on lipid content as well as bile salt. The identities of these compounds were confirmed by tandem mass spectrometric (MS/MS) experiments and comparisons with literature. CONCLUSIONS: Imaging of whole zebra fish is possible using ambient ionization techniques such as DESI. Analyses are fast and reliable. For most of the compounds observed, the identification by MS/MS can be performed directly from the fish tissue sample.