Akos Vertes

Ambient Molecular Imaging and Depth Profiling of Live Tissue by Infrared Laser Ablation Electrospray Ionization Mass Spectrometry

Mass spectrometry in conjunction with atmospheric pressure ionization methods enables the in vivo investigation of biochemical changes with high specificity and sensitivity. Laser ablation electrospray ionization (LAESI) is a recently introduced ambient ionization method suited for the analysis of biological samples with sufficient water content. With LAESI mass spectrometric analysis of chimeric Aphelandra squarrosa leaf tissue, we identify the metabolites characteristic for the green and yellow sectors of variegation. Significant parts of the related biosynthetic pathways (e.g., kaempferol biosynthesis) are ascertained from the detected metabolites and metabolomic databases. Scanning electron microscopy of the ablated areas indicates the feasibility of both two-dimensional imaging and depth profiling with a approximately 350 microm lateral and approximately 50 microm depth resolution. Molecular distributions of some endogenous metabolites show chemical contrast between the sectors of variegation and quantitative changes as the ablation reaches the epidermal and mesophyll layers. Our results demonstrate that LAESI mass spectrometry opens a new way for ambient molecular imaging and depth profiling of metabolites in biological tissues and live organisms.

In Situ Metabolic Profiling of Single Cells by Laser Ablation Electrospray Ionization Mass Spectrometry

Depending on age, phase in the cell cycle, nutrition, and environmental factors, individual cells exhibit large metabolic diversity. To explore metabolic variations in cell populations, laser ablation electrospray ionization (LAESI) mass spectrometry (MS) was used for the in situ analysis of individual cells at atmospheric pressure. Single cell ablation was achieved by delivering mid-IR laser pulses through the etched tip of a GeO(2)-based glass fiber. Metabolic analysis was performed from single cells and small cell populations of Allium cepa and Narcissus pseudonarcissus bulb epidermis, as well as single eggs of Lytechinus pictus. Of the 332 peaks detected for A. cepa, 35 were assigned to metabolites with the help of accurate ion masses and tandem MS. The metabolic profiles from single cells of the two plant species included a large variety of oligosaccharides including possibly fructans in A. cepa, and alkaloids, e.g., lycorine in N. pseudonarcissus. Analysis of adjacent individual cells with a difference in pigmentation showed that, in addition to essential metabolites found in both variants, the pigmented cells contained anthocyanidins, other flavonoids, and their glucosides. Analysis of single epidermal cells from different scale leaves in an A. cepa bulb showed metabolic differences corresponding to their age. Our results indicate the feasibility of using LAESI-MS for the in situ analysis of metabolites in single cells with potential applications in studying cell differentiation, changes due to disease states, and response to xenobiotics.

Three-Dimensional Imaging of Metabolites in Tissues under Ambient Conditions by Laser Ablation Electrospray Ionization Mass Spectrometry

Three-dimensional (3D) imaging of molecular distributions offers insight into the correlation between biochemical processes and the spatial organization of a biological tissue. Simultaneous identification of diverse molecules is a virtue of mass spectrometry (MS) that in combination with ambient ion sources enables the atmospheric pressure investigation of biomolecular distributions and processes. Here, we report on the development of an MS-based technique that allows 3D chemical imaging of tissues under ambient conditions without sample preparation. The method utilizes laser ablation electrospray ionization (LAESI) for direct molecular imaging with lateral and depth resolutions of approximately 300 microm and 30-40 microm, respectively. We demonstrate the feasibility of LAESI 3D imaging MS of metabolites in the leaf tissues of Peace lily (Spathiphyllum lynise) and the variegated Zebra plant (Aphelandra squarrosa). Extensive tandem MS studies help with the structure identification of the metabolites. The 3D distributions are found to exhibit tissue-specific metabolite accumulation patterns that correlate with the biochemical roles of these chemical species in plant defense and photosynthesis. Spatial correlation coefficients between the intensity distributions of different ions help to identify colocalization of metabolites and potentially uncover connections between metabolic pathways.

Simultaneous Imaging of Small Metabolites and Lipids in Rat Brain Tissues at Atmospheric Pressure by Laser Ablation Electrospray Ionization Mass Spectrometry

Atmospheric pressure imaging mass spectrometry is a rapidly expanding field that offers advantages in the ability to study biological systems in their native condition, simplified sample preparation, and high-throughput experiments. In laser ablation electrospray ionization (LAESI), the native water molecules in biological tissues facilitate sampling by a focused mid-infrared laser beam. The ionization of the ablated material is accomplished by electrospray postionization. In this work, we demonstrate that the imaging variant of LAESI simultaneously provides lateral distributions for small metabolites and lipids directly in rat brain sections. To cope with the fragile nature and potential dehydration of the brain tissue due to drying in the ambient environment as well as to minimize analyte redistribution, a Peltier cooling stage is integrated into the LAESI imaging system. We demonstrate the utility of high-resolution (m/Deltam > 6000) time-of-flight mass spectrometry with LAESI to deconvolute spatial distributions of different chemical species with identical nominal mass. To help with the evaluation of the massive data sets, Pearson colocalization maps are calculated for selected small metabolites and lipids. We show that this approach reveals biologically meaningful correlations between these two classes of biomolecules.

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.

The effect of the matrix on film properties in matrix-assisted pulsed laser evaporation

Thin films of polyethylene glycol of average molecular weight 1400 amu have been deposited by matrix-assisted pulsed laser evaporation (MAPLE). The deposition was carried out in vacuum (∼10−6 Torr) with an ArF (λ=193 nm) laser at a fluence of 220–230 mJ/cm2. Films were deposited on NaCl plates and glass microscope slides. Both deionized water (H2O) and chloroform (CHCl3) were used as matrices. The physiochemical properties of the films are compared via Fourier transform infrared spectroscopy, and electrospray ionization mass spectrometry. The results show that the matrix used during MAPLE can greatly affect the chemical structure and molecular weight distribution of the deposited film. The infrared absorption spectrum shows evidence for C–Cl bond formation when CHCl3 is used as a matrix, while there is little evidence in the IR data for photochemical modification when H2O is used as a matrix. Time-of-flight analysis was performed using a quadrupole mass spectrometer to monitor evaporation of a frozen CHCl...

Protein Profile of Tax-associated Complexes

Infection with human T-cell leukemia virus type 1 (HTLV-1) results in adult T-cell leukemia and HTLV-1-associated myelopathy/tropical spastic paraparesis. Tax, a 40-kDa protein, regulates viral and cellular transcription, host signal transduction, the cell cycle, and apoptosis. Tax has been shown to modulate cellular CREB and NFκB pathways; however, to date, its role in binding to various host cellular proteins involved in tumorigenesis has not been fully described. In this study, we describe the Tax-associated proteins and their functions in cells using several approaches. Tax eluted from a sizing column mostly at an apparent molecular mass of 1800 kDa. Following Tax immunoprecipitation, washes with high salt buffer, two-dimensional gel separation, and mass spectrometric analysis, a total of 32 proteins was identified. Many of these proteins belong to the signal transduction and cytoskeleton pathways and transcription/chromatin remodeling. A few of these proteins, including TXBP151, have been shown previously to bind to Tax. The interaction of Tax with small GTPase-cytoskeleton proteins, such as ras GAP1m, Rac1, Cdc42, RhoA, and gelsolin, indicates how Tax may regulate migration, invasion, and adhesion in T-cell cancers. Finally, the physical and functional association of Tax with the chromatin remodeling SWI/SNF complex was assessed using in vitro chromatin remodeling assays, chromatin remodeling factor BRG1 mutant cells, and RNA interference experiments. Collectively, Tax is able to bind and regulate many cellular proteins that regulate transcription and cytoskeletal related pathways, which might explain the pleiotropic effects of Tax leading to T-cell transformation and leukemia in HTLV-1-infected patients.

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.

Analytical Challenges of Microbial Biofilms on Medical Devices

Microbial colonization of medical devices is a widespread problem that tests the limits of conventional analytical methods. Successful analytical endeavors require collaboration between clinicians, microbiologists, biomedical engineers, and analytical chemists.

Resonant infrared pulsed-laser deposition of polymer films using a free-electron laser

Thin films of polyethylene glycol (MW 1500) have been prepared by pulsed-laser deposition (PLD) using both a tunable infrared (λ=2.9 μm, 3.4 μm) and an ultraviolet laser (λ=193 nm). A comparison of the physicochemical properties of the films by means of Fourier transform infrared spectroscopy, electrospray ionization mass spectrometry, and matrix-assisted laser desorption and ionization shows that when the IR laser is tuned to a resonant absorption in the polymer, the IR PLD thin films are identical to the starting material, whereas the UV PLD show significant structural modification. These results are important for several biomedical applications of organic and polymeric thin films.