Experimental and Theoretical Analysis of Tricyclic Antidepressants by Ultraviolet Picosecond Laser Desorption Post-Ionization Mass Spectrometry.

Imipramine class tricyclic antidepressants have low ionization efficiencies that make them difficult to detect by using secondary ion mass spectrometry. Ultraviolet picosecond laser desorption postionization (ps-LDPI-MS) is examined here for the detection of four tricyclic antidepressants: imipramine, desipramine, amitriptyline, and clomipramine. About 30 ps laser pulses at either 213 nm (5.8 eV) or 355 nm (3.5 eV) are used for desorption of samples under vacuum, 7.9 eV (157 nm) fluorine laser pulses are used for post-ionization, and the ions so formed are detected by time-of-flight mass spectrometry. Detection of imipramine by 213 nm ps-LDPI-MS shows less fragmentation than either 355 nm ps-LDPI-MS or prior results from 800 nm fs-LDPI-MS. Ionization energies of imipramine, desipramine, amitriptyline, and clomipramine are predicted using density functional theory calculations and used to explain the corresponding ps-LDPI-MS data for these four compounds as resulting from single-photon ionization. The experimental observation of low-mass amine-containing fragments with calculated ionization energies below 7.9 eV is attributed mostly to dissociation during laser desorption, followed by single-photon ionization of the neutral fragments rather than the more traditional mechanism of unimolecular dissociation following single-photon ionization of the parent molecule.

Logistic Regression Analysis of LC-MS/MS Data of Monomers Eluted from Aged Dental Composites: A Supervised Machine-Learning Approach.

Compound identification by database searching that matches experimental with library mass spectra is commonly used in mass spectrometric (MS) data analysis. Vendor software often outputs scores that represent the quality of each spectral match for the identified compounds. However, software-generated identification results can differ drastically depending on the initial search parameters. Machine learning is applied here to provide a statistical evaluation of software-generated compound identification results from experimental tandem MS data. This task was accomplished using the logistic regression algorithm to assign an identification probability value to each identified compound. Logistic regression is usually used for classification, but here it is used to generate identification probabilities without setting a threshold for classification. Liquid chromatography coupled with quadrupole-time-of-flight tandem MS was used to analyze the organic monomers leached from resin-based dental composites in a simulated oral environment. The collected tandem MS data were processed with vendor software, followed by statistical evaluation of these results using logistic regression. The assigned identification probability to each compound provides more confidence in identification beyond solely by database matching. A total of 21 distinct monomers were identified among all samples, including five intact monomers and chemical degradation products of bisphenol A glycidyl methacrylate (BisGMA), oligomers of bisphenol-A ethoxylate methacrylate (BisEMA), triethylene glycol dimethacrylate (TEGDMA), and urethane dimethacrylate (UDMA). The logistic regression model can be used to evaluate any database-matched liquid chromatography-tandem MS result by training a new model using analytical standards of compounds present in a chosen database and then generating identification probabilities for candidates from unknown data using the new model.

Femtosecond Laser Desorption Postionization MS vs ToF-SIMS Imaging for Uncovering Biomarkers Buried in Geological Samples.

The study of lipid molecular fossils by traditional biomarker analysis requires bulk sample crushing, followed by solvent extraction, and then the analysis of the extract by gas chromatography-mass spectrometry (GC-MS). This traditional analysis mixes all organic compounds in the sample regardless of their origins, with a loss of information on the spatial distribution of organic molecules within the sample. These shortcomings can be overcome using the chemical mapping of intact samples. Spectroscopic techniques such as UV fluorescence or Raman spectroscopy, laser ablation inductively coupled plasma mass spectrometry, and time-of-flight secondary ion mass spectrometry (ToF-SIMS) are among those elemental and molecular mapping techniques. This study employed femtosecond (fs) laser ablation combined with single-photon ionization, a method called fs-laser desorption postionization mass spectrometry (fs-LDPI-MS). A pulsed ∼75 fs, 800 nm laser was used to ablate the geological sample, which was then photoionized after a few microseconds by a pulsed 7.9 eV vacuum ultraviolet laser. An organic carbon-rich geological sample was used for this study to map hydrocarbon biomarkers in sediments that were previously studied by GC-MS. The petrography of this sample was examined by optical and fluorescence microscopy. It is demonstrated here that fs-LDPI-MS combined with petrography for multimodal imaging can expose buried compounds within the sample via in situ layer removal. When used in conjunction with traditional organic geochemical analysis, this method has the potential to determine the spatial distribution of organic biomarkers in geological material. Finally, fs-LDPI-MS imaging data are compared with ToF-SIMS imaging that is commonly used for such studies.

Competitive resource allocation to metabolic pathways contributes to overflow metabolisms and emergent properties in cross-feeding microbial consortia.

Resource scarcity is a common stress in nature and has a major impact on microbial physiology. This review highlights microbial acclimations to resource scarcity, focusing on resource investment strategies for chemoheterotrophs from the molecular level to the pathway level. Competitive resource allocation strategies often lead to a phenotype known as overflow metabolism; the resulting overflow byproducts can stabilize cooperative interactions in microbial communities and can lead to cross-feeding consortia. These consortia can exhibit emergent properties such as enhanced resource usage and biomass productivity. The literature distilled here draws parallels between in silico and laboratory studies and ties them together with ecological theories to better understand microbial stress responses and mutualistic consortia functioning.

Solid Sampling with a Diode Laser for Portable Ambient Mass Spectrometry.

A hand-held diode laser is implemented for solid sampling in portable ambient mass spectrometry (MS). Specifically, a pseudocontinuous wave battery-powered surgical laser diode is employed for portable laser diode thermal desorption (LDTD) at 940 nm and compared with nanosecond pulsed laser ablation at 2940 nm. Postionization is achieved in both cases using atmospheric pressure photoionization (APPI). The laser ablation atmospheric pressure photoionization (LAAPPI) and LDTD-APPI mass spectra of sage leaves (Salvia officinalis) using a field-deployable quadrupole ion trap MS display many similar ion peaks, as do the mass spectra of membrane grown biofilms of Pseudomonas aeruginosa. These results indicate that LDTD-APPI method should be useful for in-field sampling of plant and microbial communities, for example, by portable ambient MS. The feasibility of many portable MS applications is facilitated by the availability of relatively low cost, portable, battery-powered diode lasers. LDTD could also be coupled with plasma- or electrospray-based ionization for the analysis of a variety of solid samples.

High lateral resolution vs molecular preservation in near-IR fs-laser desorption postionization mass spectrometry.

Ultrashort pulse length lasers operating in the near-infrared region show promise for submicrometer lateral resolution by laser desorption-based mass spectrometry (MS) imaging. However, these experiments must balance lateral resolution and molecular fragmentation since abundant atomic ions are observed at the high laser irradiances that can be generated by tightly focused ultrashort pulse laser beams. It is shown here that combining ultrashort pulse laser desorption with laser postionization (fs-LDPI) allows for a considerable increase of molecular ion signal while operating with lower laser irradiances, yielding the added benefit of reduced molecular fragmentation. This Letter presents several experimental results in support of the fs-LDPI approach for MS imaging. First, the lateral resolution for MS imaging of molecular species desorbed by ∼75 fs, 800 nm laser pulses was determined to be <2 µm for a simulated organic electronic device under vacuum. Next, the dependence of precursor ion survival on both desorption laser fluence and delay between desorption and photoionization laser pulses was observed for a small molecule desorbed from an organic multilayer that was originally devised as a model of a bacterial biofilm. When considered in light of recent results in the literature (Milasinovic et al. J. Phys. Chem. C 2014, DOI: 10.1021/jp504062u), these experiments demonstrate the potential for submicrometer spatial resolution MS imaging by fs-LDPI.

Ion sources for mass spectrometric identification and imaging of molecular species.

Covering: 2013 The ability to transfer molecular species to the gas phase and ionize them is central to the study of natural products and other molecular species by mass spectrometry (MS). MS-based strategies in natural products have focused on a few established ion sources, such as electron impact and electrospray ionization. However, a variety of other ion sources are either currently in use to evaluate natural products or show significant future promise. This review discusses these various ion sources in the context of other articles in this special issue, but is also applicable to other fields of analysis, including materials science. Ion sources are grouped based on the current understanding of their predominant ion formation mechanisms. This broad overview groups ion sources into the following categories: electron ionization and single photon ionization; chemical ionization-like and plasma-based; electrospray ionization; and, laser desorption-based. Laser desorption-based methods are emphasized with specific examples given for laser desorption postionization sources and their use in the analysis of intact microbial biofilms. Brief consideration is given to the choice of ion source for various sample types and analyses, including MS imaging.

Dental composite biodeterioration in the presence of oral Streptococci and extracellular metabolic products.

OBJECTIVE: Secondary caries is a primary cause of early restoration failure. While primary dental caries has been extensively researched, our knowledge about the impact of secondary caries on dental restorations is relatively limited. In this study, we examined how different clinically relevant microbially-influenced environments impact the degradation of nano-filled (FIL) and micro-hybrid (AEL) dental composites. METHODS: Material strength of two commercial dental composites was measured following incubation in aqueous media containing: i) cariogenic (Streptococcus mutans) and non-cariogenic bacteria (Streptococcus sanguinis) grown on sucrose or glucose, ii) abiotic mixtures of artificial saliva and sucrose and glucose fermentation products (volatile fatty acids and ethanol) in proportions known to be produced by these microorganisms, and iii) abiotic mixtures of artificial saliva and esterase, a common oral extracellular enzyme. RESULTS: Nano-filled FIL composite strength decreased in all three types of incubations, while micro-hybrid AEL composite strength only decreased significantly in biotic incubations. The strength of both composites was statistically significantly decreased in all biotic incubations containing both cariogenic and non-cariogenic bacteria beyond that induced by either abiotic mixtures of fermentation products or esterase alone. Finally, there were no statistically significant differences in composite strength decrease among the tested biotic conditions. CONCLUSIONS: The results show that conditions created during the growth of both cariogenic and non-cariogenic oral Streptococci substantially reduce commercial composite strength, and this effect warrants further study to identify the mechanism(s). CLINICAL SIGNIFICANCE: Dental biofilms of oral Streptococci bacteria significantly affect the mechanical strength of dental restorations.

Differentiation of microbial species and strains in coculture biofilms by multivariate analysis of laser desorption postionization mass spectra.

7.87 to 10.5 eV vacuum ultraviolet (VUV) photon energies were used in laser desorption postionization mass spectrometry (LDPI-MS) to analyze biofilms comprised of binary cultures of interacting microorganisms. The effect of photon energy was examined using both tunable synchrotron and laser sources of VUV radiation. Principal components analysis (PCA) was applied to the MS data to differentiate species in Escherichia coli-Saccharomyces cerevisiae coculture biofilms. PCA of LDPI-MS also differentiated individual E. coli strains in a biofilm comprised of two interacting gene deletion strains, even though these strains differed from the wild type K-12 strain by no more than four gene deletions each out of approximately 2000 genes. PCA treatment of 7.87 eV LDPI-MS data separated the E. coli strains into three distinct groups, two "pure" groups, and a mixed region. Furthermore, the "pure" regions of the E. coli cocultures showed greater variance by PCA at 7.87 eV photon energies compared to 10.5 eV radiation. This is consistent with the expectation that the 7.87 eV photoionization selects a subset of low ionization energy analytes while 10.5 eV is more inclusive, detecting a wider range of analytes. These two VUV photon energies therefore give different spreads via PCA and their respective use in LDPI-MS constitute an additional experimental parameter to differentiate strains and species.

Laser desorption VUV postionization MS imaging of a cocultured biofilm.

Laser desorption postionization mass spectrometry (LDPI-MS) imaging is demonstrated with a 10.5 eV photon energy source for analysis and imaging of small endogenous molecules within intact biofilms. Biofilm consortia comprised of a synthetic Escherichia coli K12 coculture engineered for syntrophic metabolite exchange are grown on membranes and then used to test LDPI-MS analysis and imaging. Both E. coli strains displayed many similar peaks in LDPI-MS up to m/z 650, although some observed differences in peak intensities were consistent with the appearance of byproducts preferentially expressed by one strain. The relatively low mass resolution and accuracy of this specific LDPI-MS instrument prevented definitive assignment of species to peaks, but strategies are discussed to overcome this shortcoming. The results are also discussed in terms of desorption and ionization issues related to the use of 10.5 eV single-photon ionization, with control experiments providing additional mechanistic information. Finally, 10.5 eV LDPI-MS was able to collect ion images from intact, electrically insulating biofilms at ~100 µm spatial resolution. Spatial resolution of ~20 µm was possible, although a relatively long acquisition time resulted from the 10 Hz repetition rate of the single-photon ionization source.

Mass Spectral Imaging to Map Plant-Microbe Interactions.

Plant-microbe interactions are of rising interest in plant sustainability, biomass production, plant biology, and systems biology. These interactions have been a challenge to detect until recent advancements in mass spectrometry imaging. Plants and microbes interact in four main regions within the plant, the rhizosphere, endosphere, phyllosphere, and spermosphere. This mini review covers the challenges within investigations of plant and microbe interactions. We highlight the importance of sample preparation and comparisons among time-of-flight secondary ion mass spectroscopy (ToF-SIMS), matrix-assisted laser desorption/ionization (MALDI), laser desorption ionization (LDI/LDPI), and desorption electrospray ionization (DESI) techniques used for the analysis of these interactions. Using mass spectral imaging (MSI) to study plants and microbes offers advantages in understanding microbe and host interactions at the molecular level with single-cell and community communication information. More research utilizing MSI has emerged in the past several years. We first introduce the principles of major MSI techniques that have been employed in the research of microorganisms. An overview of proper sample preparation methods is offered as a prerequisite for successful MSI analysis. Traditionally, dried or cryogenically prepared, frozen samples have been used; however, they do not provide a true representation of the bacterial biofilms compared to living cell analysis and chemical imaging. New developments such as microfluidic devices that can be used under a vacuum are highly desirable for the application of MSI techniques, such as ToF-SIMS, because they have a subcellular spatial resolution to map and image plant and microbe interactions, including the potential to elucidate metabolic pathways and cell-to-cell interactions. Promising results due to recent MSI advancements in the past five years are selected and highlighted. The latest developments utilizing machine learning are captured as an important outlook for maximal output using MSI to study microorganisms.

The Determination of the Spatial Distribution of Indigenous Lipid Biomarkers in an Immature Jurassic Sediment Using Time-of-Flight-Secondary Ion Mass Spectrometry.

The ability to detect and map lipids, including potential lipid biomarkers, within a sedimentary matrix using mass spectrometry (MS) imaging may be critical to determine whether potential lipids detected in samples returned from Mars are indigenous to Mars or are contaminants. Here, we use gas chromatography-mass spectrometry (GC-MS) and time-of-flight-secondary ion mass spectrometry (ToF-SIMS) datasets collected from an organic-rich, thermally immature Jurassic geologic sample to constrain MS imaging analysis of indigenous lipid biomarkers in geologic samples. GC-MS data show that the extractable fractions are dominated by C27-C30 steranes and sterenes as well as isorenieratene derivatives. ToF-SIMS spectra from organic matter-rich laminae contain a strong, spatially restricted signal for ions m/z 370.3, m/z 372.3, and m/z 386.3, which we assign to C27 sterenes, cholestane (C27), and 4- or 24-methyl steranes (C28), respectively, as well as characteristic fragment ions of isorenieratene derivatives, including m/z 133.1, m/z 171.1, and m/z 237.1. We observed individual steroid spatial heterogeneity at the scale of tens to hundreds of microns. The fine-scale heterogeneity observed implies that indigenous lipid biomarkers concentrated within specific regions may be detectable via ToF-SIMS in samples with even low amounts of organic carbon, including in samples returned from Mars.

Relationships between composite roughness and Streptococcus mutans biofilm depth under shear in vitro.

OBJECTIVE: To investigate the effect of substrate, surface roughness, and hydraulic residence time (HRT) on Streptococcus mutans biofilms growing on dental composites under conditions relevant to the oral cavity. METHODS: Dental composites were prepared with varying amounts of polishing and incubated in a CDC bioreactor with an approximate shear of 0.4 Pa. S. mutans biofilms developed in the bioreactors fed sucrose or glucose and at 10-h or 40-h HRT for one week. Biofilms were characterized by confocal laser microscopy (CLM). Composite surface roughness was characterized by optical profilometry, and pre- and post-incubation composite surface fine structure and elemental composition were determined using scanning electron microscopy-energy dispersive spectroscopy (SEM-EDS). RESULTS: Polishing had a significant impact on surface roughness, varying by a factor of 15 between the polished samples and the unpolished control. S. mutans biofilms grew statistically significantly thicker on the unpolished composites. Biofilm thickness was greater at shorter 10-h HRT compared to 40-h HRT. In most cases, biofilm thickness was not statistically significantly greater in sucrose-fed bioreactors than in glucose-fed bioreactors. SEM-EDS analysis did not identify any significant change in elemental composition after aging. CONCLUSIONS: Accurate characterization of oral cavity biofilms must consider shear forces and the use of techniques that minimize alteration of the biofilm structure. Under shear, surface smoothness is the most important factor determining S. mutans biofilm thickness followed by HRT, while sucrose presence did not result in significantly greater biofilm thickness. CLINICAL SIGNIFICANCE: The obvious patterns of S. mutans growth along sub-micron scale grooving created by the polishing process suggested that initial biofilm attachment occurred in the shear-protected grooves. These results suggest that fine polishing may help prevent the initial formation of S. mutans biofilms compared to unpolished/coarse polished composites.

Stoichiometric models of sucrose and glucose fermentation by oral streptococci: Implications for free acid formation and enamel demineralization.

OBJECTIVES: The objective of this study is to develop stoichiometric models of sugar fermentation and cell biosynthesis for model cariogenic Streptococcus mutans and non-cariogenic Streptococcus sanguinis to better understand and predict metabolic product formation. METHODS: Streptococcus mutans (strain UA159) and Streptococcus sanguinis (strain DSS-10) were grown separately in bioreactors fed brain heart infusion broth supplemented with either sucrose or glucose at 37 °C. Cell mass concentration and fermentation products were measured at different hydraulic residence times (HRT) to determine cell growth yield. RESULTS: Sucrose growth yields were 0.080 ± 0.0078 g cell/g and 0.18 ± 0.031 g cell/g for S. sanguinis and S. mutans, respectively. For glucose, this reversed, with S. sanguinis having a yield of 0.10 ± 0.0080 g cell/g and S. mutans 0.053 ± 0.0064 g cell/g. Stoichiometric equations to predict free acid concentrations were developed for each test case. Results demonstrate that S. sanguinis produces more free acid at a given pH than S. mutans due to lesser cell yield and production of more acetic acid. Greater amounts of free acid were produced at the shortest HRT of 2.5 hr compared to longer HRTs for both microorganisms and substrates. SIGNIFICANCE: The finding that the non-cariogenic S. sanguinis produces greater amounts of free acids than S. mutans strongly suggests that bacterial physiology and environmental factors affecting substrate/metabolite mass transfer play a much greater role in tooth or enamel/dentin demineralization than acidogenesis. These findings enhance the understanding of fermentation production by oral streptococci and provide useful data for comparing studies under different environmental conditions.

Understanding the Influence of Li7La3Zr2O12 Nanofibers on Critical Current Density and Coulombic Efficiency in Composite Polymer Electrolytes.

Composite polymer electrolytes (CPEs) are attractive materials for solid-state lithium metal batteries, owing to their high ionic conductivity from ceramic ionic conductors and flexibility from polymer components. As with all lithium metal batteries, however, CPEs face the challenge of dendrite formation and propagation. Not only does this lower the critical current density (CCD) before cell shorting, but the uncontrolled growth of lithium deposits may limit Coulombic efficiency (CE) by creating dead lithium. Here, we present a fundamental study on how the ceramic components of CPEs influence these characteristics. CPE membranes based on poly(ethylene oxide) and lithium bis(trifluoromethanesulfonyl)imide (PEO-LiTFSI) with Li7La3Zr2O12 (LLZO) nanofibers were fabricated with industrially relevant roll-to-roll manufacturing techniques. Galvanostatic cycling with lithium symmetric cells shows that the CCD can be tripled by including 50 wt % LLZO, but half-cell cycling reveals that this comes at the cost of CE. Varying the LLZO loading shows that even a small amount of LLZO drastically lowers the CE, from 88% at 0 wt % LLZO to 77% at just 2 wt % LLZO. Mesoscale modeling reveals that the increase in CCD cannot be explained by an increase in the macroscopic or microscopic stiffness of the electrolyte; only the microstructure of the LLZO nanofibers in the PEO-LiTFSI matrix slows dendrite growth by presenting physical barriers that the dendrites must push or grow around. This tortuous lithium growth mechanism around the LLZO is corroborated with mass spectrometry imaging. This work highlights important elements to consider in the design of CPEs for high-efficiency lithium metal batteries.

Feasibility of depth profiling of animal tissue by ultrashort pulse laser ablation.

Experiments were performed to examine the feasibility of mass spectrometry (MS) depth profiling of animal tissue by ~75 fs, 800 nm laser pulses to expose underlying layers of tissue for subsequent MS analysis. Matrix assisted laser desorption ionization mass spectrometry (MALDI-MS) was used to analyze phospholipids and proteins from both intact bovine eye lens tissue and tissue ablated by ultrashort laser pulses. Laser desorption postionization mass spectrometry (LDPI-MS) with 10.5 eV single photon ionization was also used to analyze cholesterol and other small molecules in the tissue before and after laser ablation. Scanning electron microscopy was applied to examine the ablation patterns in the tissue and estimate the depth of the ablation craters. Ultrashort pulse laser ablation was found to be able to remove a layer of several tens of micrometers from the surface of eye lens tissue while leaving the underlying tissue relatively undamaged for subsequent MS analysis. MS analysis of cholesterol, phospholipids, peptides, and various unidentified species did not reveal any chemical damage caused by ultrashort pulse laser ablation for analytes smaller than ~6 kDa. However, a drop in intensity of larger protein ions was detected by MALDI-MS following laser ablation. An additional advantage was that ablated tissue displayed up to an order of magnitude higher signal intensities than intact tissue when subsequently analyzed by MS. These results support the use of ultrashort pulse laser ablation in combination with MS analysis to permit depth profiling of animal tissue.

Quantification of antibiotic in biofilm-inhibiting multilayers by 7.87 eV laser desorption postionization MS imaging.

The potential of laser desorption postionization mass spectrometry (LDPI-MS) imaging for small molecule quantification is demonstrated here. The N-methylpiperazine acetamide (MPA) of ampicillin was adsorbed into polyelectrolyte multilayer surface coatings composed of chitosan and alginate, both high molecular weight biopolymers. These MPA-ampicillin spiked multilayers were then shown to inhibit the growth of Enterococcus faecalis biofilms that play a role in early stage infection of implanted medical devices. Finally, LDPI-MS imaging using 7.87 eV single-photon ionization was found to detect MPA-ampicillin within the multilayers before and after biofilm growth with limits of quantification and detection of 0.6 and 0.3 nmol, respectively. The capabilities of LDPI-MS imaging for small molecule quantification are compared to those of MALDI-MS. Furthermore, these results indicate that 7.87 eV LDPI-MS imaging should be applicable to quantification of a range of small molecular species on a variety of complex organic and biological surfaces. Finally, while MS imaging for quantification was demonstrated here using LDPI, it is a generally useful strategy that can be applied to other methods.

Identification and imaging of peptides and proteins on Enterococcus faecalis biofilms by matrix assisted laser desorption ionization mass spectrometry.

The heptapeptide ARHPHPH was identified from biofilms and planktonic cultures of two different strains of Enterococcus faecalis, V583 and ATCC 29212, using matrix assisted laser desorption ionization mass spectrometry (MALDI-MS). ARHPHPH was also imaged at the boundary of cocultured, adjacent E. faecalis and Escherichia coli (ATCC 25922) biofilms, appearing only on the E. faecalis side. ARHPHPH was proteolyzed from κ-casein, a component in the growth media, by E. faecalis microbes. Additionally, top down and bottom up proteomic approaches were combined to identify and spatially locate multiple proteins within intact E. faecalis V583 biofilms by MALDI-MS. The resultant tandem MS data were searched against the NCBInr E. faecalis V583 database to identify thirteen cytosolic and membrane proteins which have functional association with the cell surface. Two of these proteins, enolase and GAPDH, are glycolytic enzymes known to display multiple functions in bacterial virulence in related bacterial strains. This work illustrates a powerful approach for discovering and localizing multiple peptides and proteins within intact biofilms.

Brominated tyrosine and polyelectrolyte multilayer analysis by laser desorption vacuum ultraviolet postionization and secondary ion mass spectrometry.

The small molecular analyte 3,5-dibromotyrosine (Br(2)Y) and chitosan-alginate polyelectrolyte multilayers (PEM) with and without adsorbed Br(2)Y were analyzed by laser desorption postionization-mass spectrometry (LDPI-MS). LDPI-MS using a 7.87 eV laser and tunable 8-12.5 eV synchrotron vacuum ultraviolet (VUV) radiation found that desorption of clusters from Br(2)Y films allowed detection by ≤8 eV single photon ionization. Thermal desorption and electronic structure calculations determined the ionization energy of Br(2)Y to be ~8.3 ± 0.1 eV and further indicated that the lower ionization energies of clusters permitted their detection at ≤8 eV photon energies. However, single photon ionization could only detect Br(2)Y adsorbed within PEMs when using either higher photon energies or matrix addition to the sample. All samples were also analyzed by 25 keV Bi(3)(+) secondary ion mass spectrometry (SIMS), with the negative ion spectra showing strong parent ion signal which complemented that observed by LDPI-MS. However, the negative ion SIMS appeared strongly dependent on the high electron affinity of this specific analyte and the analyte's condensed phase environment.

Spatially resolved analysis of Pseudomonas aeruginosa biofilm proteomes measured by laser ablation sample transfer.

Heterogeneity in the distribution of nutrients and oxygen gradients during biofilm growth gives rise to changes in phenotype. There has been long term interest in identifying spatial differences during biofilm development including clues that identify chemical heterogeneity. Laser ablation sample transfer (LAST) allows site-specific sampling combined with label free proteomics to distinguish radially and axially resolved proteomes for Pseudomonas aeruginosa biofilms. Specifically, differential protein abundances on oxic vs. anoxic regions of a biofilm were observed by combining LAST with bottom up proteomics. This study reveals a more active metabolism in the anoxic region of the biofilm with respect to the oxic region for this clinical strain of P. aeruginosa, despite this organism being considered an aerobe by nature. Protein abundance data related to cellular acclimations to chemical gradients include identification of glucose catabolizing proteins, high abundance of proteins from arginine and polyamine metabolism, and proteins that could also support virulence and environmental stress mediation in the anoxic region. Finally, the LAST methodology requires only a few mm2 of biofilm area to identify hundreds of proteins.