Utilizing a Robotic Sprayer for High Lateral and Mass Resolution MALDI FT-ICR MSI of Microbial Cultures

The ability to visualize biochemical interactions between microbial communities using MALDI MSI has provided tremendous insights into a variety of biological fields. Matrix application using a sieve proved to be incredibly useful, but it has many limitations that include uneven matrix coverage and limitation in the types of matrices that could be employed in studies. Recently, there has been a concerted effort to improve matrix application for studying agar plated microbial cultures, many of which utilized automated matrix sprayers. Here, we describe the usefulness of using a robotic sprayer for matrix application. The robotic sprayer has two-dimensional control over where matrix is applied, and a heated capillary that allows for rapid drying of the applied matrix. This method provided a significant increase in MALDI sensitivity over the sieve method, as demonstrated by FT-ICR MS analysis, facilitating the ability to gain higher lateral resolution MS images of Bacillus subtilis than previously reported. This method also allowed for the use of different matrices to be applied to the culture surfaces.

Open image in new window

Graphical Abstract ?

     

ISOMERS OF 3,7,11-TRIMETHYLDODECA-2,4,6,8,10- PENTAENAL (A LINEAR ANALOGUE OF RETINAL) and LOWER HOMOLOGUES IN THEIR INTERACTION WITH BOVINE OPSIN and BACTERIOOPSIN

Abstract— The all-trans and 3 mono-cir isomers of the title linear pentaenal have been prepared. Results of their interaction with bovine opsin and bacterioopsin are presented. Likewise, the negative results on two lower member homologs are described. Implications on the specificity of the binding sites are discussed.     

High Resolution MALDI Imaging Mass Spectrometry of Retinal Tissue Lipids

Matrix assisted laser desorption ionization imaging mass spectrometry (MALDI IMS) has the ability to provide an enormous amount of information on the abundances and spatial distributions of molecules within biological tissues. The rapid progress in the development of this technology significantly improves our ability to analyze smaller and smaller areas and features within tissues. The mammalian eye has evolved over millions of years to become an essential asset for survival, providing important sensory input of an organism’s surroundings. The highly complex sensory retina of the eye is comprised of numerous cell types organized into specific layers with varying dimensions, the thinnest of which is the 10 μm retinal pigment epithelium (RPE). This single cell layer and the photoreceptor layer contain the complex biochemical machinery required to convert photons of light into electrical signals that are transported to the brain by axons of retinal ganglion cells. Diseases of the retina, including age-related macular degeneration (AMD), retinitis pigmentosa, and diabetic retinopathy, occur when the functions of these cells are interrupted by molecular processes that are not fully understood. In this report, we demonstrate the use of high spatial resolution MALDI IMS and FT-ICR tandem mass spectrometry in the Abca4 ^–/– knockout mouse model of Stargardt disease, a juvenile onset form of macular degeneration. The spatial distributions and identity of lipid and retinoid metabolites are shown to be unique to specific retinal cell layers.

Salamander blue-sensitive cones lost during metamorphosis

The tiger salamander lives in shallow water with bright light in the aquatic phase, and in dim tunnels or caves in the terrestrial phase. In the aquatic phase, there are five types of photoreceptors--two types of rods and three types of cones. Our previous studies showed that the green rods and blue-sensitive cones contain the same visual pigment and have the same absorbance spectra; however, the green rods have a larger photon-catch area and thus have higher light sensitivity than the blue-sensitive cones. Here we show that after metamorphosis, the terrestrial salamander looses the blue-sensitive cones, while the density of the green rods increases. Moreover, the size of the green rod outer segments is increased in the terrestrial phase, compared to that in the aquatic phase. This switch from the blue-sensitive cones to the green rods may represent an adaptation to the dim light environment of the terrestrial phase.     

One-step photochemical attachment of NHS-terminated monolayers onto silicon surfaces and subsequent functionalization

N-Hydroxysuccinimide (NHS)-ester-terminated monolayers were covalently attached in one step onto silicon using visible light. This mild photochemical attachment, starting from omega-NHS-functionalized 1-alkenes, yields a clean and flat monolayer-modified silicon surface and allows a mild and rapid functionalization of the surface by substitution of the NHS-ester moieties with amines at room temperature. Using a combination of analytical techniques (infrared reflection absorption spectroscopy (IRRAS), extensive X-ray photoelectron spectroscopy (XPS) in combination with density functional theory calculations of the XPS chemical shifts of the carbon atoms, atomic force microscopy (AFM), and static contact angle measurements), it was shown that the NHS-ester groups were attached fully intact onto the surface. The surface reactivity of the NHS-ester moieties toward amines was qualitatively and quantitatively evaluated via the reaction with para-trifluoromethyl benzylamine and biotin hydrazide.     

Fe L-edge X-ray absorption spectroscopy of low-spin heme relative to non-heme Fe complexes: delocalization of Fe d-electrons into the porphyrin ligand

Hemes (iron porphyrins) are involved in a range of functions in biology, including electron transfer, small-molecule binding and transport, and O2 activation. The delocalization of the Fe d-electrons into the porphyrin ring and its effect on the redox chemistry and reactivity of these systems has been difficult to study by optical spectroscopies due to the dominant porphyrin pi-->pi(*) transitions, which obscure the metal center. Recently, we have developed a methodology that allows for the interpretation of the multiplet structure of Fe L-edges in terms of differential orbital covalency (i.e., differences in mixing of the d-orbitals with ligand orbitals) using a valence bond configuration interaction (VBCI) model. Applied to low-spin heme systems, this methodology allows experimental determination of the delocalization of the Fe d-electrons into the porphyrin (P) ring in terms of both P-->Fe sigma and pi-donation and Fe-->P pi back-bonding. We find that pi-donation to Fe(III) is much larger than pi back-bonding from Fe(II), indicating that a hole superexchange pathway dominates electron transfer. The implications of the results are also discussed in terms of the differences between heme and non-heme oxygen activation chemistry.     

A two-step valence tautomeric transition in a dinuclear cobalt complex

A dinuclear cobalt complex with cobalt centers bridged by a bis(dioxolene) ligand exhibits a rare two-step valence tautomeric transition.     

Subgel studies of dimyristoylphosphatidylcholine bilayers

It is known that when bilayers of some saturated phosphatidylcholines are stored for 3 or more days at approximately 0 degrees C, a lamellar subgel (Lc) phase is detected at temperatures below the pretransition by differential scanning calorimetry (DSC). However, the subgel (Lc) phase and the corresponding subtransition (Lc--> Lbeta') for dimyristoylphosphatidylcholine (DMPC) has not been clearly characterized. In this study, using the temperature jump protocol first developed by Tristram-Nagle et al. for the dipalmitoylphosphatidylcholine (DPPC) system, new and accurate data characterizing the subgel formation and subtransition of DMPC were obtained through DSC and fluorescence spectroscopy with 1,6-diphenyl-1,3,5-hexatriene (DPH). It was discovered that the formation of the DMPC subgel phase requires incubation at temperatures of -5 degrees C or lower for 2 h or more. Kinetics of the subgel formation indicate that it is a very complex process and demonstrates that the planar gel phase is merely metastable below the subtransition, and not the thermodynamically stable phase. The subgel growth of DMPC is proven to be the dehydration of the headgroup region, and the subtransition is a process in which poorly hydrated DMPC becomes hydrated.     

Protease production by Streptomyces sp. isolated from brazilian cerrado soil

Streptomyces are important microorganisms because of their capacity to produce numerous bioactive molecules. In the present work protease production, by Streptomyces sp. 594 isolated from a Brazilian Cerrado soil, was maximized by optimizing a low-cost culture medium composition (casitone and sugarcane molasses) using statistical experimental design. The final protease activity (56 U/mL) was 2.8-fold and 58-fold higher than that obtained in the beginning of this study, and in a previous work, using an actinomycete selection medium, respectively. Protease production, not growth associated, appeared to be modulated by an inducer system, whereby the C/N ratio seemed to play a significant role.