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In JoVE (2)
- Atmospheric-pressure Molecular Imaging of Biological Tissues and Biofilms by LAESI Mass Spectrometry
- Direct Analysis of Single Cells by Mass Spectrometry at Atmospheric Pressure
Other Publications (36)
- Analytical Chemistry
- Analytical Chemistry
- The Journal of Biological Chemistry
- Analytical Chemistry
- Analytical Chemistry
- The Journal of Physical Chemistry. B
- The Journal of Physical Chemistry. B
- The Journal of Physical Chemistry. B
- Analytical Chemistry
- Physical Review Letters
- Analytical Chemistry
- The Journal of Biological Chemistry
- Proteomics
- Analytical Chemistry
- Analytical Chemistry
- Physical Review. E, Statistical, Nonlinear, and Soft Matter Physics
- Analytical Chemistry
- Analytical Chemistry
- Analytical Chemistry
- The Journal of Physical Chemistry. B
- Physical Review. E, Statistical, Nonlinear, and Soft Matter Physics
- Angewandte Chemie (International Ed. in English)
- Analytical Chemistry
- Analytical Chemistry
- Analytical Chemistry
- The Analyst
- The Analyst
- Methods in Molecular Biology (Clifton, N.J.)
- PloS One
- Analytical Chemistry
- Marine Genomics
- Physical Chemistry Chemical Physics : PCCP
- Chemical Communications (Cambridge, England)
- Analytical Chemistry
- Physical Chemistry Chemical Physics : PCCP
- Analytical Chemistry
Articles by Akos Vertes in JoVE
JoVE General
Atmospheric-pressure Molecular Imaging of Biological Tissues and Biofilms by LAESI Mass Spectrometry
Department of Chemistry, George Washington University
Laser ablation electrospray ionization (LAESI) is an atmospheric-pressure ion source for mass spectrometry. In the imaging mode, a mid-infrared laser probes the distributions of molecules across a tissue section or a biofilm. This technique presents a new approach for diverse bioanalytical studies carried out under native experimental conditions.
JoVE General
Direct Analysis of Single Cells by Mass Spectrometry at Atmospheric Pressure
Department of Chemistry, George Washington University
Single cell analysis is performed by mass spectrometry on plant and animal cells at atmospheric pressure by using a sharpened optical fiber to sample the cells for laser ablation electrospray ionization (LAESI) mass spectrometry.
Other articles by Akos Vertes on PubMed
Atmospheric Pressure Matrix-assisted Laser Desorption/ionization in Transmission Geometry
In both atmospheric pressure matrix-assisted laser desorption/ionization (AP MALDI) and vacuum MALDI, the laser typically illuminates the analyte on the front side of an opaque surface (reflection geometry). Another configuration consisting of laser illumination through the sample backside (transmission geometry) has been used in conventional MALDI; however, its use and the number of reports in the literature are limited. The viability of transmission geometry with AP MALDI is demonstrated here. Such a geometry is simple to implement, eliminates the restriction for a metallic sample holder, and allows for the potential analysis of samples on their native transparent surfaces, e.g., cells or tissue sections on slides.
Internal Energy of Ions Generated by Matrix-assisted Laser Desorption/ionization
To provide an objective measure of the correlation between the internal energy content of ions generated by matrix-assisted laser desorption/ionization (MALDI) and the matrix properties, a series of well-characterized benzyl-substituted benzylpyridinium salts were used as thermometer molecules (TMs). To determine the internal energy variations of analyte ions, the survival yields of TM molecular ions were measured in three different matrixes, alpha-cyano-4-hydroxycinnamic acid (CHCA), 3,5-dimethoxy-4-hydroxycinnamic acid (sinapinic acid, SA), and 2,5-dihydroxybenzoic acid (DHB). Statistical analysis of extensive survival yield data indicated that there were discernible differences among the studied matrixes. The experimental survival yields of the TM ions were used to calculate the unimolecular decomposition rate coefficient. Corresponding theoretical reaction rate coefficients were calculated based on the Rice-Ramsperger-Kassel-Marcus (RRKM) theory for different internal energies of the TMs. The internal energies of the ions were obtained by projecting the experimental rate coefficient values onto the theoretical curves obtained by the RRKM calculations. Molecular ions of the analytes showed decreasing survival yields and consequently increasing internal energies in the three matrixes in the following order: CHCA, SA, and DHB with "cold", "intermediate", and "hot" characteristics, respectively. Qualitatively, this could be interpreted as a significant departure from earlier observations suggesting an opposite trend. The classification as hot and cold matrixes should be further qualified by accounting for the influence of laser pulse energy and the nature of the analyte. Higher laser pulse energy led to an elevated level of energy transferred to the analyte, which in turn resulted in a diminished survival yield of the analyte molecular ion. It is quite possible that the assignment of hot and cold reverses as the analyte or the laser energy changes. These findings can help predict the outcome of postsource decay experiments and clarify the concept of hot and cold matrixes in MALDI mass spectrometry.
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 NFkappaB 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.
Flexing the Electrified Meniscus: the Birth of a Jet in Electrosprays
Spraying of liquids through an electrified meniscus has become a method of choice to produce ions from large biomolecules. Using mass spectrometry, the generated ions can be analyzed to provide detailed information on their composition and structure. This technique enables high-throughput protein analysis that is a prerequisite for answering the questions presented by proteomics. In this report, Taylor cone deformations are shown to play a central role in the mechanism of electrostatic spraying. Spontaneous spray current oscillations are known to exist in most electrospray regimes and affect the stream of ions introduced into the mass spectrometer. Fast time-lapse imaging of the Taylor cone throughout its evolution indicates the presence of a nodal line and standing waves on its surface. Four phases of the cone pulsation cycle (liquid accumulation, cone formation, ejection of a jet, relaxation) are established. Based on image analysis, apex velocities, curvatures, and opening angles are determined. During jet ejection, the apex velocity and the curvature exhibit singularities. Furthermore, the pulsation frequencies of the Taylor cone deformations are determined using Fourier analysis of light refraction measurements. The oscillation frequency of the electrospray current collected by the counter electrode shows close correlation to the cone deformations, providing the first direct evidence that links spray current oscillations to Taylor cone pulsation. Thus, monitoring the oscillation frequency throughout the spraying process and adjusting the spray parameters can be used to stabilize the spray. Furthermore, synchronizing the injection of ions in time-of-flight systems with the spontaneous spray oscillations may improve the signal-to-noise ratio in the collected mass spectra.
Electrospray Diagnostics by Fourier Analysis of Current Oscillations and Fast Imaging
The different spraying modes in electrospray ionization sources exhibit large variations in their ion yield and stability. To achieve consistently optimal ion production, active control of the spray parameters is desirable. To diagnose the changes in spraying mode, the spray current and its Fourier spectrum were monitored under a wide range of conditions, that is, as a function of the spray voltage, liquid flow rate, and composition. Most Fourier spectra indicated a strong dc component, a low-frequency branch at low flow rates and applied voltages, and a high-frequency branch and their harmonics. Changing of these parameters resulted in several spraying mode changes that were reflected in the Fourier spectra of the spray current. Significant mode changes and the malformation of the Taylor cone were detected as peak shifts or sudden changes in the spectrum quality. This was confirmed by fast imaging that showed a reduction in the size of the Taylor cone under hydrophobic tip conditions and rapid periodic ejection of filaments and droplets for high conductivity solutions. Comparing the oscillation frequencies of Taylor cones of different sizes, good correlation was found with the frequencies of capillary waves on comparablly sized liquid spheres. Spray stability was also linked with the positional stability of the contact line between the liquid meniscus and the capillary tip.
Surface Modification and Laser Pulse Length Effects on Internal Energy Transfer in DIOS
Benzyl-substituted benzylpyridinium (BP) chloride salts were used as a source of thermometer ions to probe the internal energy (IE) transfer in desorption/ionization on porous silicon (DIOS). To modify their wetting properties and the interaction energies with the thermometer ions, the DIOS surfaces were silylated to produce trimethylsilyl- (TMS), amine- (NH2), perfluoroalkyl- (PFA), and perfluorophenyl-derivatized (PFP) surfaces. Two laser sources--a nitrogen laser with pulse length of 4 ns and a mode locked 3 x omega Nd:YAG laser with a pulse length of 22 ps--were utilized to induce desorption/ionization and fragmentation at various laser fluence levels. The corresponding survival yields were determined as indicators of the IE transfer and the IE distributions were extracted. In most cases, with increasing the laser fluence in a broad range (approximately 20 mJ/cm2), no change in IE transfer was observed. For ns excitation, this was in remarkable contrast with MALDI, where increasing the laser fluence resulted in sharply (within approximately 5 mJ/cm2) declining survival yields. Derivatization of the porous silicon surface did not affect the survival yields significantly but had a discernible effect on the threshold fluence for ion production. The IE distributions determined for DIOS and MALDI from alpha-cyano-4-hydroxycinnamic acid reveal that the mean IE value is always lower for the latter. Using the ps laser, the IE distribution is always narrower for DIOS, whereas for ns laser excitation the width depends on surface modification. Most of the differences between MALDI and DIOS described here are compatible with the different dimensionality of the plume expansion and the differences in the activation energy of desorption due to surface modifications.
Charge Reduction in Electrosprays: Slender Nanojets As Intermediates
Molecular dynamics simulations were used to study charge reduction in electrosprayed liquids through the formation of slender nanojet intermediates. The dynamics of shape relaxation and disintegration were followed as a function of charge in cylindrical water nanojets containing protonated diglycine molecules. Depending on the overall charge, simulations showed three basic scenarios for nanojet evolution. Moderately charged nanojets reduced to spheres, whereas nanojets charged close to the Rayleigh limit divided into two offspring droplets. Due to the large Coulomb interaction between ions, highly charged nanojets suffered repeated fission until the resulting droplets were charged below the Rayleigh limit. We demonstrated the role of surface fluctuations and Maxwell stress distributions in the disintegration process. The relaxation dynamics of the moderately charged systems to spherical geometry followed a damped oscillator behavior. Compared to neutral water jets, the presence of charges in subcritical nanojets resulted in a stiffer system with longer relaxation times to spherical geometry. Interparticle forces acting between the separating offspring droplets in nanojet breakup were also determined. Due to the increased role of fluctuations in nanojets, the Rayleigh limit was shown to overestimate the maximum charge on stable systems indicating higher nanodroplet production efficiency than one would expect from macroscopic theories alone.
Internal Energy Transfer in Laser Desorption/ionization from Silicon Nanowires
Laser-induced desorption/ionization from silicon nanowires (SiNW) is an emerging method for mass spectrometry of small to medium-size molecules. In this new technique, we examined the internal energy transfer to seven benzylpyridinium thermometer ions and extracted the corresponding internal energy distributions. To explore the effect of the energy-deposition rate on the internal energy transfer, two lasers with significantly different pulse lengths (4 ns vs 22 ps) were utilized as excitation sources. A comparison of ion yields indicated that the SiNW substrates required 5-8 times less laser fluence for ion production than either matrix-assisted laser desorption/ionization (MALDI) or desorption/ionization on silicon (DIOS). In contrast however, the survival yield (SY) values showed that the internal energy transferred to the thermometer ions was more than (ps laser) or comparable to (ns laser) MALDI but it was significantly less than in DIOS. The internal energy transfer was only slightly dependent on laser fluence and on wire density. These effects were rationalized in terms of the confinement of thermal energy in the nanowires and of unimpeded three-dimensional plume expansion. Unlike in MALDI from CHCA and in perfluorophenyl-derivatized DIOS, for desorption from SiNWs the effect of laser pulse length on the internal energy transfer was found to be negligible.
Adjustable Fragmentation in Laser Desorption/ionization from Laser-induced Silicon Microcolumn Arrays
Laser-induced silicon microcolumn arrays (LISMA) were developed as matrix-free substrates for soft laser desorption/ionization mass spectrometry (SLDI-MS). When low-resistivity silicon wafers were irradiated in air, sulfur hexafluoride, or water environment with multiple pulses from a 3 x omega mode-locked Nd:YAG laser, columnar structures were formed on the surface. The radii of curvature of the column tips varied with the processing environment, ranging from approximately 120 nm in water, to <1 mum in SF6, and to approximately 2 mum in air. In turn, these microcolumn arrays were used as matrix-free soft laser desorption substrates. In SLDI-MS experiments with a nitrogen laser, the microcolumn arrays obtained in water environment readily produced molecular ions for peptides and synthetic polymers at low laser fluence. These surfaces demonstrated the best ion yield among the three arrays. The threshold laser fluence and ion yield were comparable to those observed in matrix-assisted laser desorption/ionization. Low-femtomole sensitivity and approximately 6000 Da mass range were achieved. At elevated laser fluence, efficient in-source decay was observed and extensive peptide sequence information was extracted from the resulting mass spectra. The versatility of LISMA was attributed to confinement effects due to the submicrometer morphology and to the surface, thermal, and optical properties of processed silicon.
Order-chaos-order Transitions in Electrosprays: the Electrified Dripping Faucet
Electrosprays have diverse applications including protein analysis, electrospinning, and nanoencapsulation for drug delivery. We show that a variety of electrospray regimes exhibit fundamental analogy with the nonlinear dynamics of a dripping faucet. The applied voltage in electrosprays results in additional period doublings and temporal order-chaos-order transitions. Attractors in the return maps show logarithmic self-similarity in time, suggesting self-similar capillary waves on the meniscus. The bifurcations in ejection time can be explained by phase variations between capillary waves and pinch-off conditions and by ejection mode changes due to contact angle variations.
Atmospheric Pressure Molecular Imaging by Infrared MALDI Mass Spectrometry
An atmospheric pressure (AP) MALDI imaging interface was developed for an orthogonal acceleration time-of-flight mass spectrometer and utilized to analyze peptides, carbohydrates, and other small biomolecules using infrared laser excitation. In molecular imaging experiments, the spatial distribution of mock peptide patterns was recovered with a detection limit of approximately 1 fmol/pixel from a variety of MALDI matrixes. With the use of oversampling for the image acquisition, a spatial resolution of 40 microm, 5 times smaller than the laser spot size, was achieved. This approach, however, required that the analyte was largely removed at the point of analysis before the next point was interrogated. Native water in plant tissue was demonstrated to be an efficient natural matrix for AP infrared laser desorption ionization. In soft fruit tissues from bananas, grapes, and strawberries, potassiated ions of the most abundant metabolites, small carbohydrates, and their clusters produced the strongest peaks in the spectra. Molecular imaging of a strawberry skin sample revealed the distribution of the sucrose, glucose/fructose, and citric acid species around the embedded seeds. Infrared AP MALDI mass spectrometric imaging without the addition of an artificial matrix enables the in vivo investigation of small biomolecules and biological processes (e.g., metabolomics) in their natural environment.
Identifying the Membrane Proteome of HIV-1 Latently Infected Cells
Profiling integral plasma membrane proteins is of particular importance for the identification of new biomarkers for diagnosis and for drug development. We report in this study the identification of surface markers by performing comparative proteomics of established human immunodeficiency virus-1 (HIV-1) latent cell models and parental cell lines. To this end we isolated integral membrane proteins using a biotin-directed affinity purification method. Isolated proteins were separated by two-dimensional gel electrophoresis and identified by matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) after in gel digestion. Seventeen different proteins were found to vary on the surface of T-cells due to HIV-1 infection. Of these proteins, 47% were integral membrane proteins, and 18% were membrane-associated. Through the use of complementary techniques such as Western blotting and fluorescent staining, we confirmed the differential expression of some of the proteins identified by MALDI-TOF including Bruton's tyrosine kinase and X-linked inhibitor of apoptosis. Finally, using phosphatidylinositol 3-kinase inhibitors and flavopiridol to inhibit Bruton's tyrosine kinase localization at the membrane and X-linked inhibitor of apoptosis protein expression, respectively, we showed that HIV-1 latently infected cells are more sensitive to these drugs than uninfected cells. This suggests that HIV-1 latently infected cells may be targeted with drugs that alter several pathways that are essential for the establishment and maintenance of latency.
The Proteome Survey of an Electricity-generating Organ (Torpedo Californica Electric Organ)
Torpedo californica is a species in class Chondrichthyes. Electric rays have evolved the electric organ, which is similar to the mammalian neuromuscular junction (NMJ). Here, we took a combined cDNA sequencing and proteomic approach to define the molecular constituents of the T. californica electric organ. For soluble proteins, 2-DE was used and 224 protein spots were mapped. Plasma membrane fractions were analyzed using the shotgun approach (LC-MS/MS). A Torpedo cDNA library was constructed and 607 cDNA clones were sequenced. Identification of electric organ proteins was done using cross-species comparisons, and a custom database was constructed from cDNA translations. We unambiguously identified 121 proteins and transcripts, 103 of which were novel additions to the existing databases of Torpedo fish. Fifteen proteins of known function, but not previously associated with either the electroplaque or NMJ, were present at high abundance. These included the heat shock and oxidative stress proteins, annexin V (calelectrin), and plectin 1. Most interesting were the unambiguous matches to 11 human ORFs of unknown function, including four potential RNA splicing proteins, a vacuolar sorting protein, and a tetraspanin containing protein. This analysis identified proteins that may play a role in the higher vertebrate neuromuscular junction or other electrical synapses.
Spraying Mode Effect on Droplet Formation and Ion Chemistry in Electrosprays
Depending on the spraying conditions and fluid properties, a variety of electrospray regimes exists. Here we explore the changes in ion production that accompany the transitions among the three axial spraying modes, the burst mode, the pulsating Taylor cone mode, and the cone-jet mode. Spray current oscillation and phase Doppler anemometry measurements, fast imaging of the electrified meniscus, and mass spectrometry are utilized to study the formation, size, velocity, and chemical composition of droplets produced in the three modes. High-speed images indicate that the primary droplets are produced by varicose waves and lateral kink instabilities on the liquid jet emerging from the Taylor cone, whereas secondary droplets are formed by fission. Dramatic changes in the droplet size distributions result from the various production and breakup mechanisms observed at different emitter voltages and liquid flow rates. We demonstrate that droplet fission can be facilitated by space charge effects along the liquid jet and in the plume. Compared to the other two regimes, a significantly enhanced signal-to-noise ratio, a lower degree of analyte oxidation, and milder fragmentation are observed for the cone-jet mode.
Laser Ablation Electrospray Ionization for Atmospheric Pressure, in Vivo, and Imaging Mass Spectrometry
Mass spectrometric analysis of biomolecules under ambient conditions promises to enable the in vivo investigation of diverse biochemical changes in organisms with high specificity. Here we report on a novel combination of infrared laser ablation with electrospray ionization (LAESI) as an ambient ion source for mass spectrometry. As a result of the interactions between the ablation plume and the spray, LAESI accomplishes electrospray-like ionization. Without any sample preparation or pretreatment, this technique was capable of detecting a variety of molecular classes and size ranges (up to 66 kDa) with a detection limit of 8 and 25 fmol for verapamil and reserpine, respectively, and quantitation capabilities with a four-decade dynamic range. We demonstrated the utility of LAESI in a broad variety of applications ranging from plant biology to clinical analysis. Proteins, lipids, and metabolites were identified, and antihistamine excretion was followed via the direct analysis of bodily fluids (urine, blood, and serum). We also performed in vivo spatial profiling (on leaf, stem, and root) of metabolites in a French marigold (Tagetes patula) seedling.
Astable Regime in Electrosprays
Astable regimes are common in nonlinear systems ranging from electrooptic devices to cardiac rhythms under environmental stress. Electrosprays exhibit three main axial regimes (dripping, pulsating, and cone-jet). It is generally accepted that the transition between the pulsating and cone-jet regimes is sudden, accompanied by an abruptly enhanced spray current. Here we present an alternative to this scenario, in which the electrospray follows a new chaotic astable path to the cone-jet regime. The astable regime could be explained as a result of transitions between a limit cycle (pulsating regime) and a fixed point (cone-jet regime) in a subcritical Andronov-Hopf bifurcation due to small external perturbations (noise). With the introduction of this regime, a broader view of the diverse axial regimes becomes possible based on nonlinear dynamics that enables their consistent classification.
Conformational and Noncovalent Complexation Changes in Proteins During Electrospray Ionization
Electrospray ion sources efficiently produce gas-phase ions from proteins and their noncovalent complexes. Charge-state distributions of these ions are increasingly used to gauge their conformations in the solution phase. Here we investigate how this correlation is affected by the spraying conditions at the early stage of droplet generation, prior to the ionization process. We followed the folding behavior of model proteins cytochrome c and ubiquitin and the dissociation of the noncovalent holomyoglobin complex. Spray current measurements, fast Taylor cone imaging, and mass analysis of the generated ions indicated that the protein structure experienced conformational or complexation changes upon variations in the spraying mode of the electrospray ionization source. These effects resulted in a departure from the original secondary, tertiary, and quaternary structure of proteins, possibly introducing artifacts in related studies. Therefore, if a particular gas-phase ion conformation is required or correlations with the liquid-phase conformations are studied, it is advantageous to maintain a particular spraying mode. Alternatively, spraying mode-induced changes can be utilized to alter the structure of proteins in, for example, time-resolved experiments for the study of protein folding dynamics.
Atmospheric Pressure Infrared MALDI Imaging Mass Spectrometry for Plant Metabolomics
The utility of atmospheric pressure infrared MALDI mass spectrometry (AP IR-MALDI) was assessed for plant metabolomics studies. Tissue sections from plant organs, including flowers, ovaries, aggregate fruits, fruits, leaves, tubers, bulbs, and seeds were studied in both positive and negative ion modes. For leaves, single laser pulses sampled the cuticle and upper epidermal cells, whereas multiple pulses were demonstrated to ablate some mesophyll layers. Tandem mass spectra were obtained with collision-activated dissociation to aid with the identification of some observed ions. In the positive mode, most ions were produced as potassium, proton, or sometimes sodium ion adducts, whereas proton loss was dominant in the negative ion mode. Over 50 small metabolites and various lipids were detected in the spectra including, for example, 7 of the 10 intermediates in the citric acid cycle. Key components of the glycolysis pathway occurring in the plant cytosol were found along with intermediates of phospholipid biosynthesis and reactants or products of amino acid, nucleotide, oligosaccharide, and flavonoid biosynthesis. AP IR-MALDI mass spectrometry was used to follow the fluid transport driven by transpiration and image the spatial distributions of several metabolites in a white lily (Lilium candidum) flower petal.
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.
Competing Ion Decomposition Channels in Matrix-assisted Laser Desorption Ionization
We gauged the internal energy transfer for two dissociative ion decomposition channels in matrix-assisted laser desorption ionization (MALDI) using the benzyltriphenylphosphonium (BTP) thermometer ion [PhCH 2PPh 3] (+). Common MALDI matrixes [alpha-cyano-4-hydroxycinnamic acid (CHCA), 3,5-dimethoxy-4-hydroxycinnamic acid (sinapinic acid, SA), and 2,5-dihydroxycinnamic acid (DHB)] were studied with nitrogen laser (4 ns pulse length) and mode-locked 3 x omega Nd:YAG laser (22 ps pulse length) excitation. Despite the higher fluence required to initiate fragmentation, BTP ions indicated lower internal energy transfer with the picosecond laser in all three matrixes. These differences can be rationalized in terms of phase explosion induced by the nanosecond laser vs a stress-confinement-driven desorption mechanism for the picosecond laser. For the two ion production channels of the BTP thermometer ion, breaking a single bond can result in the formation of benzyl/tropylium ions, F1, or triphenylphosphine ions, F2. In SA and DHB, as well as in CHCA at low fluence levels, the efficiency of these channels (expressed by the branching ratio I F1/ I F2) is moderately in favor of producing tropylium ions, 1 < I F1/ I F2 < 6. As the laser fluence is increased, for CHCA, there is a dramatic shift in favor of the tropylium ion production, with I F1/ I F2 approximately 30 for the nanosecond and the picosecond laser, respectively. This change is correlated with the sudden increase in the BTP internal energies in CHCA in the same laser fluence range. The large changes observed in internal energy deposition for CHCA with laser fluence can account for its ability to induce fragmentation in peptides more readily than SA and DHB.
Early Plume Expansion in Atmospheric Pressure Midinfrared Laser Ablation of Water-rich Targets
We have developed a one-dimensional fluid dynamics model for the ablation of water-rich targets by nanosecond infrared laser pulses at atmospheric pressure. To describe the laser-target interaction and the plume expansion dynamics, in light of recent experimental results the model incorporates phase explosion due to superheating and the nonlinear light absorption properties of water. In the model, the phase explosion is treated as a prolonged process that lasts for a finite time. Once a thin layer beneath the target surface exceeds the phase explosion temperature, this layer is transformed from target material into a mixture of water vapor and droplets and become part of the plume. This process is sustained for some time until the laser energy cannot maintain it. The simulation results show that as a result of two different phase transition mechanisms, i.e., surface evaporation and phase explosion, a first, slower plume expansion phase is followed by a more vigorous accelerated expansion phase. The calculated time evolution of the shock front at various fluence levels agrees well with the experimental observations of Apitz and Vogel [I. Apitz and A. Vogel, Appl. Phys. A. 81, 329 (2005)]. This model sheds light on the effect of phase explosion in laser ablation dynamics and its results are relevant for material synthesis, surface analysis, and medical (surgery) applications.
Nanophotonic Ion Production from Silicon Microcolumn Arrays
Nanoantennas for ions: Silicon microcolumn arrays harvest light from a laser pulse to produce ions. The system behaves like a quasi-periodic antenna array with ion yields that show profound dependence on the plane of polarization and the angle of incidence of the laser beam. Photonic ion sources promise to enable enhanced control of ion production on a micro- and nanometer scale and direct integration with miniaturized analytical devices.
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.
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.
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.
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.
Laser Ablation Electrospray Ionization for Atmospheric Pressure Molecular Imaging Mass Spectrometry
Laser ablation electrospray ionization (LAESI) is a novel method for the direct imaging of biological tissues by mass spectrometry. By performing ionization in the ambient environment, this technique enables in vivo studies with potential for single-cell analysis. A unique aspect of LAESI mass spectrometric imaging (MSI) is depth profiling that, in combination with lateral imaging, permits 3D molecular imaging for the first time under native conditions. With current lateral and depth resolutions of approximately 100 and approximately 40 microm, respectively, LAESI MSI helps to explore the molecular architecture of live tissues.
Direct Detection of Diverse Metabolic Changes in Virally Transformed and Tax-expressing Cells by Mass Spectrometry
Viral transformation of a cell starts at the genetic level, followed by changes in the proteome and the metabolome of the host. There is limited information on the broad metabolic changes in HTLV transformed cells.
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.
Evolution and Comparative Genomics of Subcellular Specializations: EST Sequencing of Torpedo Electric Organ
Uncharacterized open reading frames (ORFs) in human genomic sequence often show a high degree of evolutionary conservation, yet have little or no tissue EST or protein data suggestive of protein product function. The encoded proteins may have highly restricted expression in specialized cells, subcellular specializations, and/or narrow windows during development. One such highly specialized and minute subcellular compartment is the neuromuscular junction (NMJ), where motorneurons contact muscle fibers. The electric Torpedo ray has evolved to expand the NMJ structure to the size of a large organ (electroplax organ), and we hypothesized that Torpedo electroplax proteins would be candidates for human ESTs expressed at the human NMJ. A total of 9719 primary electroplax cDNA clones were sequenced. We identified 44 human ORFs showing high (>63%) amino acid identity to Torpedo electroplax transcripts with enrichment for mRNA splicing motifs (SH2 and pre-mRNA splicing domains), an observation potentially important for the strict nuclear domains maintained by myonuclei underlying the NMJ. We generated antibodies against two uncharacterized human genes (C19orf29 [Drosophila cactin] and C15orf24) and showed that these were indeed expressed at the murine NMJ. Cactin, a member of the Rel transcription factor family in Drosophila, localized to the postsynaptic cytosol of the NMJ and nuclear membrane. C15orf24 protein localized to the murine postsynaptic sarcolemma. We show a novel approach towards identifying proteins expressed at a subcellular specialization using evolutionary diversity of organ function and cross-species mapping.
Polarization Dependent Fragmentation of Ions Produced by Laser Desorption from Nanopost Arrays
Tailored silicon nanopost arrays (NAPA) enable controlled and resonant ion production in laser desorption ionization experiments and have been termed nanophotonic ion sources (Walker et al., J. Phys. Chem. C, 2010, 114, 4835-4840). As the post dimensions are comparable to or smaller than the laser wavelength, near-field effects and localized electromagnetic fields are present in their vicinity. In this contribution, we explore the desorption and ionization mechanism by studying how surface derivatization affects ion yields and fragmentation. We demonstrate that by increasing the laser fluence on derivatized NAPA with less polar surfaces that have decreased interaction energy between the structured silicon substrate and the adsorbate, the spectrum changes from exhibiting primarily molecular ions to showing a growing variety and abundance of fragments. The polarization angle of the laser beam had been shown to dramatically affect the ion yields of adsorbates. For the first time, we report that by rotating the plane of polarization of the desorption laser, the internal energy of the adsorbate can also be modulated resulting in polarization dependent fragmentation. This polarization effect also resulted in selective fragmentation of vitamin B(12). To explore the internal energy of NAPA generated ions, the effect of the post aspect ratios on the laser desorption thresholds and on the internal energy of a preformed ion was studied. Elevated surface temperatures and enhanced near fields in the vicinity of high aspect ratio posts are thought to contribute to desorption and ionization from NAPA. Comparison of the fluence dependence of the internal energies of ions produced from nanoporous silicon and NAPA substrates indicates that surface restructuring or transient melting by the desorption laser is a prerequisite for the former but not for the latter.
Rapid, Non-targeted Discovery of Biochemical Transformation and Biomarker Candidates in Oncovirus-infected Cell Lines Using LAESI Mass Spectrometry
Finding insights into how viruses hijack metabolic processes and biomarkers for viral diseases often require hypotheses about target compounds and/or labelling techniques. Here we present a method based on laser ablation electrospray ionization mass spectrometry to rapidly identify potential protein and metabolite biomarkers of oncovirus infection in B lymphocytes.
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.
Internal Energy Deposition and Ion Fragmentation in Atmospheric-pressure Mid-infrared Laser Ablation Electrospray Ionization
Mid-infrared laser ablation of water-rich targets at the maximum of the 2.94 μm absorption band is a two-step process initiated by phase explosion followed by recoil pressure induced material ejection. Particulates and/or droplets ejected by this high temperature high pressure process can be ionized for mass spectrometry by charged droplets from an electrospray. In order to gauge the internal energy introduced in this laser ablation electrospray ionization (LAESI®) process, we apply the survival yield method and compare the results with electrospray ionization (ESI) and matrix-assisted laser desorption ionization (MALDI). The results indicate that LAESI yields ions with internal energies indistinguishable from those produced by ESI. This finding is consistent with the recoil pressure induced ejection of low micrometre droplets that does not significantly change the internal energy of solute molecules.
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.
