Desorption Electrospray Ionization Cyclic Ion Mobility-Mass Spectrometry Imaging for Traumatic Brain Injury Spatial Metabolomics.

Lipidomics focuses on investigating alterations in a wide variety of lipids that harness important information on metabolic processes and disease pathology. However, the vast structural diversity of lipids and the presence of isobaric and isomeric species creates serious challenges in feature identification, particularly in mass spectrometry imaging experiments that lack front-end separations. Ion mobility has emerged as a potential solution to address some of these challenges and is increasingly being utilized as part of mass spectrometry imaging platforms. Here, we present the results of a pilot mass spectrometry imaging study on rat brains subjected to traumatic brain injury (TBI) to evaluate the depth and quality of the information yielded by desorption electrospray ionization cyclic ion mobility mass spectrometry (DESI cIM MSI). Imaging data were collected with one and six passes through the cIM cell. Increasing the number of passes increased the ion mobility resolving power and the resolution of isobaric lipids, enabling the creation of more specific maps. Interestingly, drift time data enabled the recognition of multiply charged phosphoinositide species in the complex data set generated. These species have not been previously reported in TBI MSI studies and were found to decrease in the hippocampus region following injury. These changes were attributed to increased enzymatic activity after TBI, releasing arachidonic acid that is converted to eicosanoids to control inflammation. A substantial reduction in NAD and alterations in other adenine metabolites were also observed, supporting the hypothesis that energy metabolism in the brain is severely disrupted in TBI.

Feature-based molecular networking in the GNPS analysis environment.

Molecular networking has become a key method to visualize and annotate the chemical space in non-targeted mass spectrometry data. We present feature-based molecular networking (FBMN) as an analysis method in the Global Natural Products Social Molecular Networking (GNPS) infrastructure that builds on chromatographic feature detection and alignment tools. FBMN enables quantitative analysis and resolution of isomers, including from ion mobility spectrometry.

Direct Drug Analysis in Polymeric Implants Using Desorption Electrospray Ionization - Mass Spectrometry Imaging (DESI-MSI).

PURPOSE: Desorption electrospray ionization mass spectrometry imaging (DESI-MSI) coupled with gas-phase ion mobility spectrometry was used to characterize the drug distribution in polymeric implants before and after exposure to accelerated in vitro release (IVR) media. DESI-MSI provides definitive chemical identification and localization of formulation components, including 2D chemical mapping of individual components with essentially no sample preparation. METHODS: Polymeric implants containing 40% (w/w) entecavir and poly(D,L-lactide) (PLA) were prepared and then exposed to either acidified PBS (pH 2.5) or MeOH:H2O (50:50, v/v) medias during a 7-day IVR test using continuous flow-through (CFT) cell dissolution. The amount of drug released from the polymer matrix during the 7-day IVR test was monitored by online-ultraviolet spectroscopy (UV) and HPLC-UV. After that period, intact implants and radial sections of implants were analyzed by DESI-MSI with ion mobility spectrometry. The active ingredient along with impurities and contaminants were used to generate chemical maps before and after exposure to the release medias. RESULTS: Bi-phasic release profiles were observed for implants during IVR release using both medias. During the second phase of release, implants exposed to PBS, pH 2.5, released the entecavir faster than the implants exposed to MeOH:H2O (50:50, v/v). Radial images of the polymer interior show that entecavir is localized along the central core of the implant after exposure to MeOH:H2O (50:50, v/v) and that the drug is more uniformly distributed throughout the implant after exposure to acidified PBS (pH 2.5). CONCLUSIONS: DESI-MSI coupled with ion mobility analysis produced chemical images of the drug distribution on the exterior and interior of cylindrical polymeric implants before and after exposure to various release medias. These results demonstrated the utility of this technique for rapid characterization of drug and impurity/degradant distribution within polymeric implants with direct implications for formulation development as well as analytical method development activities for various solid parenteral and oral dosage forms. These results are especially meaningful since samples were analyzed with essentially no preparative procedures.

Human T-lymphotropic virus type 1-infected cells secrete exosomes that contain Tax protein.

Human T-lymphotropic virus type 1 (HTLV-1) is the causative agent of adult T-cell leukemia and HTLV-1-associated myelopathy/tropical spastic paraparesis. The HTLV-1 transactivator protein Tax controls many critical cellular pathways, including host cell DNA damage response mechanisms, cell cycle progression, and apoptosis. Extracellular vesicles called exosomes play critical roles during pathogenic viral infections as delivery vehicles for host and viral components, including proteins, mRNA, and microRNA. We hypothesized that exosomes derived from HTLV-1-infected cells contain unique host and viral proteins that may contribute to HTLV-1-induced pathogenesis. We found exosomes derived from infected cells to contain Tax protein and proinflammatory mediators as well as viral mRNA transcripts, including Tax, HBZ, and Env. Furthermore, we observed that exosomes released from HTLV-1-infected Tax-expressing cells contributed to enhanced survival of exosome-recipient cells when treated with Fas antibody. This survival was cFLIP-dependent, with Tax showing induction of NF-κB in exosome-recipient cells. Finally, IL-2-dependent CTLL-2 cells that received Tax-containing exosomes were protected from apoptosis through activation of AKT. Similar experiments with primary cultures showed protection and survival of peripheral blood mononuclear cells even in the absence of phytohemagglutinin/IL-2. Surviving cells contained more phosphorylated Rb, consistent with the role of Tax in regulation of the cell cycle. Collectively, these results suggest that exosomes may play an important role in extracellular delivery of functional HTLV-1 proteins and mRNA to recipient cells.

In Situ Analysis of Small Populations of Adherent Mammalian Cells Using Laser Ablation Electrospray Ionization Mass Spectrometry in Transmission Geometry.

Most cultured cells used for biomedical research are cultured adherently, and the requisite detachment prior to biochemical analysis might induce chemical changes. This is especially crucial if accurate metabolic measurements are desired, given the rapid turnover of metabolites in living organisms. There are only a few methods available for the nontargeted in situ analysis of small adherent cell populations. Here we show that laser ablation electrospray ionization (LAESI) mass spectrometry (MS) can be used to analyze adherent cells directly, while still attached to the culture surface. To reduce the size of the analyzed cell population, the spot size constraints of conventional focusing in reflection geometry (rg) LAESI had to be eliminated. By introducing transmission geometry (tg) LAESI and incorporating an objective with a high numerical aperture, spot sizes of 10-20 µm were readily achieved. As few as five adherent cells could be specifically selected for analysis in their culturing environment. The importance of in situ analysis was highlighted by comparing the metabolite composition of adherent versus suspended cells. For example, we observed that cells analyzed adherently yielded higher values for the adenylate energy charge (0.90 ± 0.09 for adherent cells vs 0.09 ± 0.03 for suspended cells). Additionally, due to the smaller focal spot size, tg-LAESI enabled the analysis of ∼20 times smaller cell populations compared to rg-LAESI.

High-throughput cell and tissue analysis with enhanced molecular coverage by laser ablation electrospray ionization mass spectrometry using ion mobility separation.

Ambient ionization methods, such as laser ablation electrospray ionization (LAESI), facilitate the direct analysis of unperturbed cells and tissues in their native states. However, the lack of a separation step in these ionization techniques results in limited molecular coverage due to interferences, ion suppression effects, and the lack of ability to differentiate between structural isomers and isobaric species. In this contribution, LAESI mass spectrometry (MS) coupled with ion mobility separation (IMS) is utilized for the direct analysis of protein mixtures, megakaryoblast cell pellets, mouse brain sections, and Arabidopsis thaliana leaves. We demonstrate that the collision cross sections of ions generated by LAESI are similar to the ones obtained by ESI. In various applications, LAESI-IMS-MS allows for the high-throughput separation and mass spectrometric detection of biomolecules on the millisecond time scale with enhanced molecular coverage. For example, direct analysis of mouse brain tissue without IMS had yielded ∼300 ionic species, whereas with IMS over 1 100 different ions were detected. Differentiating between ions of similar mass-to-charge ratios with dissimilar drift times in complex biological samples removes some systematic distortions in isotope distribution patterns and improves the fidelity of molecular identification. Coupling IMS with LAESI-MS also expands the dynamic range by increasing the signal-to-noise ratio due to the separation of isobaric or other interfering ionic species. We have also shown that identification of potential biomarkers by LAESI can be enhanced by using the drift times of individual ions as an additional parameter in supervised orthogonal projections to latent structures discriminant analysis. Comparative analysis of drift time versus mass-to-charge ratio plots was performed for similar tissue samples to pinpoint significant metabolic differences.

Laser ablation atmospheric pressure photoionization mass spectrometry imaging of phytochemicals from sage leaves.

RATIONALE: Despite fast advances in ambient mass spectrometry imaging (MSI), the study of neutral and nonpolar compounds directly from biological matrices remains challenging. In this contribution, we explore the feasibility of laser ablation atmospheric pressure photoionization (LAAPPI) for MSI of phytochemicals in sage (Salvia officinalis) leaves. METHODS: Sage leaves were studied by LAAPPI-time-of-flight (TOF)-MSI without any sample preparation. Leaf mass spectra were also recorded with laser ablation electrospray ionization (LAESI) mass spectrometry and the spectra were compared with those obtained by LAAPPI. RESULTS: Direct probing of the plant tissue by LAAPPI efficiently produced ions from plant metabolites, including neutral and nonpolar terpenes that do not have polar functional groups, as well as oxygenated terpene derivatives. Monoterpenes and monoterpenoids could also be studied from sage by LAESI, but only LAAPPI was able to detect larger nonpolar compounds, such as sesquiterpenes and triterpenoid derivatives, from the leaf matrix. Alternative MSI methods for nonpolar compounds, such as desorption atmospheric pressure photoionization (DAPPI), do not achieve as good spatial resolution as LAAPPI (<400 µm). CONCLUSIONS: We show that MSI with LAAPPI is a useful tool for concurrently studying the distribution of polar and nonpolar compounds, such as phytochemicals, directly from complex biological samples, and it can provide information that is not available by other, established methods.

Metabolic transformation of microalgae due to light acclimation and genetic modifications followed by laser ablation electrospray ionization mass spectrometry with ion mobility separation.

Metabolic profiling of various microalga species and their genetic variants, grown under varied environmental conditions, has become critical to accelerate the exploration of phytoplankton biodiversity and biology. The accumulation of valuable metabolites, such as glycerolipids, is also sought in microalgae for biotechnological applications ranging from food, feed, medicine, cosmetics to bioenergy and green chemistry. In this report we describe the direct analysis of metabolites and lipids in small cell populations of the green alga Chlamydomonas reinhardtii, using laser ablation electrospray ionization (LAESI) mass spectrometry (MS) coupled with ion mobility separation (IMS). These microorganisms are capable of redirecting energy storage pathways from starch to neutral lipids depending on environmental conditions and nutrient availability. Metabolite and lipid productions were monitored in wild type (WT), and genetically modified C. reinhardtii strains with an impaired starch pathway. Lipids, such as triacylglycerols (TAG) and diacylglyceryl-N,N,N-trimethylhomoserine (DGTS), were monitored over time under altered light conditions. More than 200 ions related to metabolites, e.g., arginine, cysteine, serine, palmitate, chlorophyll a, chlorophyll b, etc., were detected. The lipid profiles at different light intensities for strains with impaired starch pathway (Sta1 and Sta6) contained 26 glycerolipids, such as DGTS, monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG), as well as 33 TAG species. Results were obtained over a 72 hour time period under high and low light conditions for the WT species and the two mutants. Our results indicate that LAESI-IMS-MS can be utilized for the rapid analysis of increased TAG production at elevated light intensities. Compared to WT, the Sta6 strain showed 2.5 times higher lipid production at 72 hours under high light conditions. The results demonstrate our ability to rapidly observe numerous changes in metabolite and lipid levels in microalgal population. These capabilities are expected to facilitate the exploration of genetically altered microalgal strains for biofuel production.

In situ metabolic analysis of single plant cells by capillary microsampling and electrospray ionization mass spectrometry with ion mobility separation.

Advances in single cell analysis techniques have demonstrated cell-to-cell variability in both homogeneous and heterogeneous cell populations strengthening our understanding of multicellular organisms and individual cell behaviour. However, additional tools are needed for non-targeted metabolic analysis of live single cells in their native environment. Here, we combine capillary microsampling with electrospray ionization (ESI) mass spectrometry (MS) and ion mobility separation (IMS) for the analysis of various single A. thaliana epidermal cell types, including pavement and basal cells, and trichomes. To achieve microsampling of different cell types with distinct morphology, custom-tailored microcapillaries were used to extract the cell contents. To eliminate the isobaric interferences and enhance the ion coverage in single cell analysis, a rapid separation technique, IMS, was introduced that retained ions based on their collision cross sections. For each cell type, the extracted cell material was directly electrosprayed resulting in ∼200 peaks in ESI-MS and ∼400 different ions in ESI-IMS-MS, the latter representing a significantly enhanced coverage. Based on their accurate masses and tandem MS, 23 metabolites and lipids were tentatively identified. Our results indicated that profound metabolic differences existed between the trichome and the other two cell types but differences between pavement and basal cells were hard to discern. The spectra indicated that in all three A. thaliana cell types the phenylpropanoid metabolism pathway had high coverage. In addition, metabolites from the subpathway, sinapic acid ester biosynthesis, were more abundant in single pavement and basal cells, whereas compounds from the kaempferol glycoside biosynthesis pathway were present at significantly higher level in trichomes. Our results demonstrate that capillary microsampling coupled with ESI-IMS-MS captures metabolic differences between A. thaliana epidermal cell types, paving the way for the non-targeted analysis of single plant cells and subcellular compartments.

Spatial analysis of renal acetaminophen metabolism and its modulation by 4-methylpyrazole with DESI mass spectrometry imaging.

Acute kidney injury (AKI) is a common complication in acetaminophen (APAP) overdose patients and can negatively impact prognosis. Unfortunately, N-acetylcysteine, which is the standard of care for the treatment of APAP hepatotoxicity does not prevent APAP-induced AKI. We have previously demonstrated the renal metabolism of APAP and identified fomepizole (4-methylpyrazole, 4MP) as a therapeutic option to prevent APAP-induced nephrotoxicity. However, the kidney has several functionally distinct regions, and the dose-dependent effects of APAP on renal response and regional specificity of APAP metabolism are unknown. These aspects were examined in this study using C57BL/6J mice treated with 300-1200 mg/kg APAP and mass spectrometry imaging (MSI) to provide spatial cues relevant to APAP metabolism and the effects of 4MP. We find that renal APAP metabolism and generation of the nonoxidative (APAP-GLUC and APAP-SULF) and oxidative metabolites (APAP-GSH, APAP-CYS, and APAP-NAC) were dose-dependently increased in the kidney. This was recapitulated on MSI which revealed that APAP overdose causes an accumulation of APAP and APAP GLUC in the inner medulla and APAP-CYS in the outer medulla of the kidney. APAP-GSH, APAP-NAC, and APAP-SULF were localized mainly to the outer medulla and the cortex where CYP2E1 expression was evident. Interestingly, APAP also induced a redistribution of reduced GSH, with an increase in oxidized GSH within the kidney cortex. 4MP ameliorated these region-specific variations in the formation of APAP metabolites in renal tissue sections. In conclusion, APAP metabolism has a distinct regional distribution within the kidney, the understanding of which provides insight into downstream mechanisms of APAP-induced nephrotoxicity.

Simultaneous detection of nonpolar and polar compounds by heat-assisted laser ablation electrospray ionization mass spectrometry.

A heat-assisted laser ablation electrospray ionization (HA-LAESI) method for the simultaneous mass spectrometric analysis of nonpolar and polar analytes was developed. The sample was introduced using mid-infrared laser ablation of a water-rich target. The ablated analytes were ionized with an electrospray plume, which was intercepted by a heated nitrogen gas jet that enhanced the ionization of analytes of low polarity. The feasibility of HA-LAESI was tested by analyzing, e.g., naphtho[2,3-a]pyrene, cholesterol, tricaprylin, 1,1',2,2'-tetramyristoyl cardiolipin, bradykinin fragment 1-8, and 1-palmitoyl-2-oleoyl-sn-glycerol. HA-LAESI was found better suited for low polarity compounds than conventional LAESI, whereas polar compounds were observed with both techniques. The sensitivity of HA-LAESI for the polar bradykinin fragment 1-8 was slightly lower than observed for LAESI. HA-LAESI showed a linear response for 500 nM to 1.0 mM solutions (n = 11) of verapamil with R(2) = 0.988. HA-LAESI was applied for the direct analysis of tissue samples, e.g., avocado (Persea americana) mesocarp and mouse brain tissue sections. Spectra of the avocado showed abundant triglyceride ion peaks, and the results for the mouse brain sections showed cholesterol as the main species. Conventional LAESI shows significantly lower ionization efficiency for these neutral lipids. HA-LAESI can be applied to the analysis of nonpolar and polar analytes, and it extends the capabilities of conventional LAESI to nonpolar and neutral compounds.

Comparative local analysis of metabolites, lipids and proteins in intact fish tissues by LAESI mass spectrometry.

Direct mass spectrometric analysis of animal tissues is an emerging field enabled by recent developments in ambient ion sources. Label-free in situ analysis of metabolites, lipids, and peptides/proteins from intact tissues in whole fish specimens of different gender and age were performed by laser ablation electrospray ionization (LAESI) mass spectrometry (MS). Hypertrophied glandular tissue (gill gland) of adult male Aphyocharax anisitsi (bloodfin tetra) was compared with gill tissues in females of the same species. Comparison of a large number of sample-specific ions was aided by a multivariate statistical method based on orthogonal projections to latent structures discriminant analysis. More than 200 different ions were detected in the mass spectra corresponding to primary metabolites, hormones, lipids and peptides/proteins. The gill tissues of the sexually mature males exhibited multiply charged ions in the 6+ to 10+ charge states corresponding to a protein with a molecular weight of 11 380 Da. This protein was present only in the mature male gill glands but absent in the corresponding area of the female and immature male specimens. An additional nine proteins were detected by LAESI-MS in both the male and female gill tissues.

Direct analysis of phycobilisomal antenna proteins and metabolites in small cyanobacterial populations by laser ablation electrospray ionization mass spectrometry.

Due to their significance in energy and environmental and natural product research, as well as their large genetic diversity, rapid in situ analysis of cyanobacteria is of increasing interest. Metabolic profiles and the composition of energy harvesting antenna protein complexes are needed to understand how environmental factors affect the functioning of these microorganisms. Here, we show that laser ablation electrospray ionization (LAESI) mass spectrometry enables the direct analysis of phycobilisomal antenna proteins and report on numerous metabolites from intact cyanobacteria. Small populations (n < 616 ± 76) of vegetative Anabaena sp. PCC7120 cyanobacterial cells are analyzed by LAESI mass spectrometry. The spectra reveal the ratio of phycocyanin (C-PC) and allophycocyanin (APC) in the antenna complex, the subunit composition of the phycobiliproteins, and the tentative identity of over 30 metabolites and lipids. Metabolites are tentatively identified by accurate mass measurements, isotope distribution patterns, and literature searches. The rapid simultaneous analysis of abundant proteins and diverse metabolites enables the evaluation of the environmental response and metabolic adaptation of cyanobacteria and other microorganisms.

Infrared laser ablation atmospheric pressure photoionization mass spectrometry.

In this paper we introduce laser ablation atmospheric pressure photoionization (LAAPPI), a novel atmospheric pressure ion source for mass spectrometry. In LAAPPI the analytes are ablated from water-rich solid samples or from aqueous solutions with an infrared (IR) laser running at 2.94 µm wavelength. Approximately 12 mm above the sample surface, the ablation plume is intercepted with an orthogonal hot solvent (e.g., toluene or anisole) jet, which is generated by a heated nebulizer microchip and directed toward the mass spectrometer inlet. The ablated analytes are desolvated and ionized in the gas-phase by atmospheric pressure photoionization using a 10 eV vacuum ultraviolet krypton discharge lamp. The effect of operational parameters and spray solvent on the performance of LAAPPI is studied. LAAPPI offers ~300 µm lateral resolution comparable to, e.g., matrix-assisted laser desorption ionization. In addition to polar compounds, LAAPPI efficiently ionizes neutral and nonpolar compounds. The bioanalytical application of the method is demonstrated by the direct LAAPPI analysis of rat brain tissue sections and sour orange (Citrus aurantium) leaves.

Redox phospholipidomics analysis reveals specific oxidized phospholipids and regions in the diabetic mouse kidney.

While it is generally accepted that oxidative stress impacts the diabetic kidney and contributes to pathogenesis, there is a substantial lack of knowledge about the molecular entity and anatomic location of a variety of reactive species. Here we provide a novel "oxidative stress map" of the diabetic kidney - the first of its kind, and identify specific, oxidized and other reactive lipids and their location. We used the db/db mouse model and Desorption Electrospray Ionization (DESI) mass spectrometry combined with heatmap image analysis. We analyzed a comprehensive array of phospholipid peroxide species in normal (db/m) and diabetic (db/db) kidneys using DESI imaging. Oxilipidomics heatmaps of the kidneys were generated focusing on phospholipids and their potential peroxidized products. We identified those lipids that undergo peroxidation in diabetic nephropathy. Several phospholipid peroxides and their spatial distribution were identified that were specific to the diabetic kidney, with significant enrichment in oxygenated phosphatidylethanolamines (PE) and lysophosphatidylethanolamine. Beyond qualitative and semi-quantitative information about the targets, the approach also reveals the anatomic location and the extent of lipid peroxide signal propagation across the kidney. Our approach provides novel, in-depth information of the location and molecular entity of reactive lipids in an organ with a very heterogeneous landscape. Many of these reactive lipids have been previously linked to programmed cell death mechanisms. Thus, the findings may be relevant to understand what impact phospholipid peroxidation has on cell and mitochondria membrane integrity and redox lipid signaling in diabetic nephropathy.

Desorption Electrospray Ionization Mass Spectrometry Imaging Allows Spatial Localization of Changes in Acetaminophen Metabolism in the Liver after Intervention with 4-Methylpyrazole.

Acetaminophen (APAP) overdose is the most common cause of acute liver failure in the US, and hepatotoxicity is initiated by a reactive metabolite which induces characteristic centrilobular necrosis. The only clinically available antidote is N-acetylcysteine, which has limited efficacy, and we have identified 4-methylpyrazole (4MP, Fomepizole) as a strong alternate therapeutic option, protecting against generation and downstream effects of the cytotoxic reactive metabolite in the clinically relevant C57BL/6J mouse model and in humans. However, despite the regionally restricted necrosis after APAP, our earlier studies on APAP metabolites in biofluids or whole tissue homogenate lack the spatial information needed to understand region-specific consequences of reactive metabolite formation after APAP overdose. Thus, to gain insight into the regional variation in APAP metabolism and study the influence of 4MP, we established a desorption electrospray ionization mass spectrometry imaging (DESI-MSI) platform for generation of ion images for APAP and its metabolites under ambient air, without chemical labeling or a prior coating of tissue which reduces chemical interference and perturbation of small molecule tissue localization. The spatial intensity and distribution of both oxidative and nonoxidative APAP metabolites were determined from mouse liver sections after a range of APAP overdoses. Importantly, exclusive differential signal intensities in metabolite abundance were noted in the tissue microenvironment, and 4MP treatment substantially influenced this topographical distribution.

In situ cell-by-cell imaging and analysis of small cell populations by mass spectrometry.

Molecular imaging by mass spectrometry (MS) is emerging as a tool to determine the distribution of proteins, lipids, and metabolites in tissues. The existing imaging methods, however, mostly rely on predefined rectangular grids for sampling that ignore the natural cellular organization of the tissue. Here we demonstrate that laser ablation electrospray ionization (LAESI) MS can be utilized for in situ cell-by-cell imaging of plant tissues. The cell-by-cell molecular image of the metabolite cyanidin, the ion responsible for purple pigmentation in onion (Allium cepa) epidermal cells, correlated well with the color of cells in the tissue. Chemical imaging using single-cells as voxels reflects the spatial distribution of biochemical differences within a tissue without the distortion stemming from sampling multiple cells within the laser focal spot. Microsampling by laser ablation also has the benefit of enabling the analysis of very small cell populations for biochemical heterogeneity. For example, with a ∼30 µm ablation spot we were able to analyze 3-4 achlorophyllous cells within an oil gland on a sour orange (Citrus aurantium) leaf. To explore cell-to-cell variations within and between tissues, multivariate statistical analysis on LAESI-MS data from epidermal cells of an A. cepa bulb and a C. aurantium leaf and from human buccal epithelial cell populations was performed using the method of orthogonal projections to latent structures discriminant analysis (OPLS-DA). The OPLS-DA analysis of mass spectra, containing over 300 peaks each, provided guidance in identifying a small number of metabolites most responsible for the variance between the cell populations. These metabolites can be viewed as promising candidates for biomarkers that, however, require further verification.

Direct analysis of lipids and small metabolites in mouse brain tissue by AP IR-MALDI and reactive LAESI mass spectrometry.

Ambient analysis of metabolites and lipids from unprocessed animal tissue by mass spectrometry remains a challenge. The utility of the two novel ambient ionization techniques--atmospheric pressure infrared matrix-assisted laser desorption ionization (AP IR-MALDI) and laser ablation electrospray ionization (LAESI)--is demonstrated for the direct mass spectrometric analysis of lipids and other metabolites from mouse brain. Major brain lipids including cholesterol, various phospholipid species (glycerophosphocholines, sphingomyelin and phosphatidylethanolamines) along with numerous metabolites, for example g-aminobutyric acid (GABA), creatine and choline, were identified in a typical mass spectrum. In a new ionization modality of LAESI, termed reactive LAESI, in-plume reactions with a solute of choice (lithium sulfate) enhanced structure-specific fragmentation of lipid ions for improved molecular assignment in collision-activated dissociation experiments. In-plume processes in reactive LAESI provide additional structural information without contaminating the biological sample with the reactant.

Minimally invasive monitoring of cellulose degradation by desorption electrospray ionization and laser ablation electrospray ionization mass spectrometry.

Minimally invasive desorption electrospray ionization-mass spectrometry (DESI-MS) and laser ablation electrospray ionization-MS (LAESI-MS) were used to look for soluble cellulose degradation products produced by accelerated aging in unsized cotton paper. Soluble extracts from papers aged 144 to 26,856 hours were first analyzed in solution using traditional electrospray ionization-MS (ESI-MS). Results were compared to those from direct analysis of condensed phase degradation products extracted from the absorbent paper substrate using DESI-MS and LAESI-MS. ESI-MS results showed evidence of oligosaccharide degradation products ranging from cellobiose to cellononaose; using DESI-MS and LAESI-MS, products from cellobiose to cellodecaose and glucose to cellooctaose, respectively, were observed. As degradation proceeded, increased quantities of both low and high molecular weight oligosaccharides were observed. The analytical approaches developed in the control study were applied for the detection of degradation products in two naturally-aged books dating from the 19th century, both made from cotton and linen. Oligosaccharides ranging from glucose to cellopentaose were observed.

Single-Cell Metabolomics by Mass Spectrometry.

Single-cell level metabolomics gives a snapshot of small molecules, intermediates, and products of cellular metabolism within a biological system. These small molecules, typically less than 1 kDa in molecular weight, often provide the basis of biochemical heterogeneity within cells. The molecular differences between cells with a cell type are often attributed to random stochastic biochemical processes, cell cycle stages, environmental stress, and diseased states. In this chapter, current limitations and challenges in single-cell analysis by mass spectrometry will be discussed alongside the prospects of single-cell metabolomics in systems biology. A few selected example of the recent development in mass spectrometry tools to unravel single-cell metabolomics will be described as well.