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Minute Papers Due 09/07/2012

Please post this week's minute papers as "comments" to this post. Minute papers should be posted by 5 pm on Friday (though they may not appear on the blog immediately). Feel free to read your classmate's posts.

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Title: Pressure-Assisted Capillary Electrophoresis Coupling with Matrix-Assisted Laser Desorption/Ionization-Mass Spectrometric Imaging for Quantitative Analysis of Complex Peptide Mixtures

Journal: Analytical Chemistry

Coupling separation and detection methods has become an area of interest as it usually results in a time reduction and an increase in resolution. This article describes a new separation-detection technique for pressure-assisted capillary electrophoresis-mass spectrometric imaging (PACE-MSI). In this technique, capillary electrophoresis (CE) is directly coupled to a matrix assisted laser desorption/ionization (MALDI) plate for introduction into a time-of-flight (TOF) mass analyzer.

The authors used a CE setup that was made to directly deposit along a line on a MALDI plate under ambient conditions. By raising the initial CE buffer beaker above the outlet of the capillary to the MALDI plate, partial pressure driven flow was induced and allowed for even and precise flow control at the CE outlet for deposition. This could be controlled by the initial beaker height and separation voltage. The outgoing CE flow from the separation was interfaced to a MALDI plate where a co-matrix was simultaneously deposited at a 1:1 ratio of CE sample to co-matrix in the presence of a heat lamp. This interface was done without sheath-flow by the use of a fracture in the outlet end of the capillary that was covered in a membrane allowing for a narrower CE trace on the MALDI plate. The direct coupling of CE and MALDI imaging along with the new interface and addition of the siphon driving force allowed for better resolution than the “typical” method for offline MALDI coupling with CE fraction collection, which greatly reduces peak capacities achieved in a separation. Also, an upgrade to MALDI imaging versus a manually operated MALDI that was used in previous work allowed for better resolution and reduced time with automatic control of the MALDI stage.

The authors successfully developed and implemented the separation and improved detection of peptides by CE coupled to temporal resolved detection by MALDI-TOF/TOF, but there are some areas that could be explored further. With the two liquid phases mixing and then drying on the plate there is dilution of the sample and the possibility of mixing after deposition depending on how fast the solution dries, which the authors did not address thoroughly. Perhaps by optimization of the co-matrix introduction and sample drying, even better peak capacity and signal would be possible. If the authors tried introducing the co-matrix at higher concentration and lower volumes, reducing the dilution, the signal might be increased. They could also try coating the plate with matrix instead of introducing it in stream. As for diffusion, the authors could modify their setup slightly, adding a slow nitrogen flow around the capillary and heat the MALDI plate or heat the solution just before it comes out of the capillary. This could de-solvate the CE buffer quicker, reducing on-plate diffusion, resulting in even better resolution and more detectable peptides. Optimization experiments would need to be conducted to find the best way to de-solvate the sample such that it dries just as it exits the capillary and touches the MALDI plate, and not before.

Title: In Vivo Characterization of Atherosclerotic Plaque Depositions by Raman-Probe Spectroscopy and in Vitro Coherent Anti-Stokes Raman Scattering Microscopic Imaging on a Rabbit Model

Journal: Analytical Chemistry

Plaque, material consisting primarily of fat and cholesterol, can accumulate inside arteries and restrict blood flow. This condition is known as atherosclerosis and is a prominent health concern among Western society. There is much interest in gaining more knowledge regarding varying plaque composition and location throughout the human body in order to determine effective treatment techniques. Positron emission tomography, magnetic resonance imaging, intravascular ultrasound and optical coherence tomography have all been performed in past studies; however issues with low sensitivity and invasive procedures prevent the use of these techniques from becoming widespread. Raman and infrared spectroscopy has been employed in past experiments as well, including several in vivo studies in which the chemical composition of plaque was determined. The authors in this paper attempt to report on the chemical composition of varying plaque deposits in vivo using a Raman spectroscopy probe as well as compare results of traditional Raman spectroscopy to those performed by Coherent anti-Stokes Raman scattering (CARS) which operates on a shorter time scale.

Raman spectroscopy experiments proceeded via a Raman probe with a 100 μm diameter enclosed within a steel tube at 1 mm diameter. A total of eighteen experiments were conducted, sixteen in vitro and two in vivo, at a wavelength of 785 nm. During the CARS imaging an anti-Stokes signal of approximately 561 nm was collected and detected via a photomultiplier tube. All in vivo studies were performed on adult male white rabbits that were given a 0.5% diet of cholesterol for varying amounts of time.

In vivo experiments concluded mostly protein features were present in the distal aorta, thoracic aorta and aortic arch while the ascending aorta contained little protein and primarily lipid characteristics. These lipid features were the result of triglycerides. Comparison studies using in vitro models confirmed in vivo results however the overall spectral quality was improved due to the absence of hemoglobin Raman scattering. Normal aorta and plaque-deposited aorta samples were examined using CARS but direct recording was not possible due to the wavelength dependence of the Stokes beam. Imaging using CARS was able to successfully distinguish between the normal and plaque-deposited tissue.

All experiments within this paper were on the time scale of one to ten seconds and contained no fluorescence, both of which are advantages for further in vivo studies. Plaque depositions were detected in several regions of the aorta with lower aorta sections corresponding to decreased plaque depositions. Overall compositions of these plaque amounts were found to change as well. In vitro studies confirmed all in vivo results and produced slightly cleaner spectra. Only two in vivo studies were conducted and since plaque composition can vary from animal to animal, I believe more in vivo studies should be performed to gain a better data sampling for averaging the plaque chemical composition. Plaque composition can vary based on factors such as age and nutrition therefore I would also conduct studies examining each of these scenarios individually to gain a fuller understanding of factors influencing plaque development.

Title: Graphene Oxide Wrapping on Squaraine-Loaded Mesoporous Silica Nanoparticles for Bioimaging

By: S. Sivaramapanicker et al.

Journal: Journal of the American Chemical Society

In this publication, the authors work to resolve a common problem with in vitro fluorescence imaging via squaraine dyes. Although squaraine dyes provide excellent photophysical properties, two of their major limitations for in vitro imaging are their inclination to aggregate in aqueous environments and their susceptibility to nucleophilic by thiol-containing amino acids such as cysteine (Cys) and glutathione (GSH). These interactions negate squaraine’s adsorption and emission properties. To remedy this issue, squaraine dyes were loaded into mesoporous silica nanoparticles (MSN); these loaded MSN were then coated with graphene oxide (GO) in order to block dye access to the external environment. The methods used by the authors included nanoparticle synthesis, a host of materials characterization techniques, as well as FT-IR spectroscopy, UV-vis spectroscopy, fluorescent spectroscopy, Raman spectroscopy, and fluorescence microscopy.

As shown in Figure 2d, Raman spectra comparisons of plain MSN versus GO coated squaraine loaded MSN (GO-MSNS) suggest an effective coating procedure. The spectral band distinct to MSN is maintained in both samples, but the GO-MSNS have two characteristic GO bands. According to Figure 3a, UV-vis spectra comparisons of dye loaded and non-loaded GO coated MSN demonstrate not only the presence of the dye within the particle core, but also the accessibility of visible light to the particle core. The emission spectra (Figure 3b) further demonstrate squaraine dyes are loadable and maintained in the GO coating of MSN. Figure 4a and 4b (adsorption and emission spectra, respectively) compare treatments of free dye and GO-MSNS in the presence of either Cys or GSH over approximately 70 minutes. In the presence of Cys and GSH, free squaraine adsorption and emission time profiles deteriorate rapidly overtime, which is indicative of a loss in photophysical features. It is very apparent that squaraine dyes are maintained within the GO coated MSN core and also protected from Cys or GSH nucleophilic attack, which thereby preserves dye integrity. Treatment of HeLa cancer cells with GO-MSNS, as shown in Figure 5, was observed via epifluorescence microscopy, which illustrated the effectiveness of GO-MSNS to serve as intracellular and cell-surface bioimaging markers.

Although the paper does a fine job translating synthesis and materials characterization to a valid function, a few questions remain. The paper mentions GO-MSNS are stable in aqueous solutions for long periods of time, yet it provides no analytical study of particle integrity in relevant biological media such as simulated body fluid. Also, the supporting information provides a MTT cytotoxicity study, which suggests GO-MSNS are not likely the most viable bioimaging markers for living cells. Work concerning modification of the GO surface with biologically suitable materials should be done with the intent of improving cellular viability while maintaining the benefits of GO-MSNS. Given the robust nature of GO and its characteristic carboxylic acid functional groups, stability in simulated body fluid and modification of the GO surface should be possible.

Title: Fluorescence Imaging of Cellular Metabolites with RNA
By:Paige, J.S. et al.
Journal: Science

In this article, the authors attempt to improve the real time imaging of small molecules in cells by generalizing the sensors used to fluorescently label a molecule of interest. The analytical technique used in this article is fluorescence imaging.

In this article, the authors present a novel way to fluorescently label S¬-adenosylmethionine (SAM) in Escherichia coli. They use an RNA aptamer and Spinach, another aptamer, to bind to SAM. When they combine and attach to SAM, this causes a conformational change in the aptamer making it more stable and susceptible to fluorescing. In figure 1b, you can see the emission spectra of the RNA and Spinach aptamer. Fluorescence when bound to SAM is much, much greater than when it’s not bound to SAM. This is important because typically, a genetically encoded fluorescent protein is required to bind to the target molecule. These are less generalizable for any molecule of interest. With this new technique, any molecule of interest can theoretically be imaged in real time faster and more efficiently.

This paper is exciting and opens many doors for research of new molecules in cells. The authors claim that the design of sensors to image any molecule is possible with this technique, but I would like to see more experiments performed where other molecules are imaged with this technique. Another study I would be interested in is a binding constant study of the RNA aptamer to other molecules of interest. I would also like to know how to address the long activation time for the target molecule to actually start fluorescing significantly. Another interesting experiment would be to study a molecule in vivo, to see if real time imaging is possible using this technique. Lastly, if in vivo studies are to be performed, this can greatly affect how you record your measurements.

Title: Direct trace-elemental analysis of urine samples by laser ablation - inductively coupled plasma - mass spectrometry after sample deposition on clinical filter papers
By: M. Aramendia et al.
Journal: Analytical Chemistry

In this paper, the authors propose a new method for analyzing urine samples dried on clinical filter papers to find the concentration of various elemental metallic species. The technique used for this was Laser Ablation Inductively Coupled Plasma Mass Spectrometry (LA-ICP-MS). Advantages to collecting samples as Dried Urine Spots (DUS) on filter paper instead of as the natural solution include easier transport, cheaper long-term storage and the possibility of unsupervised sample collection by the patient instead of in-clinic collection. Challenges include low sample concentration on the filter paper and non-homogeneous analyte concentration across the filter paper.

The authors propose using small, pre-cut disks of Whatman no. 903 filter paper instead of the large sheets of paper used in similar ‘dried spot’ techniques that are used for the collection of blood samples. The smaller, more absorbent disks do not allow the sample to spread as far, allowing for higher sample concentration. Analyte concentration was tested by LA-ICP-MS. The LA-ICP-MS method was calibrated by matrix-matched calibration curves and the addition of Pt to samples as an internal standard.

The analyte distribution across the paper disk was tested by repeated LA-ICP-MS experiments. It was found that all analytes showed a higher concentration towards the disk rim, with homogeneous concentrations across a large area in the center of the disk. All later ablations were carried out on the center of the disk to avoid this chromatographic enrichment of analyte concentration on the disk rim.

The authors propose that future experiments could be carried out with lasers with higher repetition rates. This would cause the ablation of larger amounts of sample, allowing for the use of less sensitive but more commonly available methods of analysis, like quadrupole mass spectrometry.

Although the authors do not discuss the possibility, they could also try analyzing the paper disk samples with other analytical techniques which would work on analytes that their LA-ICP-MS method is not designed to work on, e.g. non-volatile organics and salts.

Title: Protein–Glycosphingolipid Interactions Revealed Using Catch-and-Release Mass Spectrometry
By: John S. Klassen
Journal: Analytical Chemistry

Lipids and proteins interact in many ways within the cells to form complexes which have essential duties in cells. “Glycosphingolipids (GSL) on the surface of cells are important receptors in antigen/microbial recognition and cell adhesion.”1 In this paper, the author’s goal is to investigate the interaction of GSL with specific proteins which can be used in many treatment developments. The techniques used in this work are: catch and release electrospray ionization mass spectrometry, collision-induced dissociation, and ion mobility separation.
Recently, electrospray ionization mass spectrometry has been applied to explore lipid-protein interactions in aqueous solutions; however, in this case the ESI-MS is not capable of analyzing the interactions between water-soluble proteins and insoluble glycosphingolipids. Thus, a novel technique known as catch and release electrospray ionization mass spectrometry CaR-ESI-MS has been used to detect these protein-ligand complexes. Cholera toxin (CT) and Shiga toxin type 2 (Stx2) are the two proteins which were used in this paper with their native GSL receptors (GM1 and GB3) respectively. In this method nanodiscs (ND) “which made up entirely of lipids extracted from cell cultures”2 have been employed as a carrier to solubilize the receptor (GSL) for numerous fundamental studies. Then the assay was prepared by mixing the glycosphingolipid-nanodisc complex with the solution containing soluble proteins. The resulting assay was transferred to the gas phase by ESI. The authors implied that the Direct ESI-MS of the protein-GSL-ND is not upfront. Thus, collision-induced dissociation (CID) method has been used to allow the detachment of the phospholipid bonds between protein-receptor and nanodisc. Following this cleavage, the remaining isolated protein-GSL complex bonds have been broken by another step of CID. Finally the mass analysis of generated fragments in conjunction with ion mobility separation (IMS) which is useful method when isomers of GSL are present employed to identify receptors. This method provides many useful outcomes. For example, they found no evidence of interactions between CT or Stx2 with their non-native GSL in their ESI mass spectra which reveals that proteins just have certain interactions with their native GSL.
Finally, they claimed that CaR-ESI-MS can be employed for detecting special protein-glycosphingolipid interactions and the possibility of using this strategy to discover unknown receptors from a complex mixture. However; there is still some work needed to be done for further discoveries about the application of this method to evaluate relative attractions for different GSL. As a complimentary I thought that electron spin resonance (ESR) and ultraviolet resonance Raman spectroscopy (UVRR) might be useful to investigate the protein-receptor bindings and the selectivity of the interactions between proteins and lipids.

Reference:
1, 2- These are come directly from this paper.

Title: Protein–Glycosphingolipid Interactions Revealed Using Catch-and-Release Mass Spectrometry
By: John S. Klassen
Journal: Analytical Chemistry

Lipids and proteins interact in many ways within the cells to form complexes which have essential duties in cells. “Glycosphingolipids (GSL) on the surface of cells are important receptors in antigen/microbial recognition and cell adhesion.”1 In this paper, the author’s goal is to investigate the interaction of GSL with specific proteins which can be used in many treatment developments. The techniques used in this work are: catch and release electrospray ionization mass spectrometry, collision-induced dissociation, and ion mobility separation.
Recently, electrospray ionization mass spectrometry has been applied to explore lipid-protein interactions in aqueous solutions; however, in this case the ESI-MS is not capable of analyzing the interactions between water-soluble proteins and insoluble glycosphingolipids. Thus, a novel technique known as catch and release electrospray ionization mass spectrometry CaR-ESI-MS has been used to detect these protein-ligand complexes. Cholera toxin (CT) and Shiga toxin type 2 (Stx2) are the two proteins which were used in this paper with their native GSL receptors (GM1 and GB3) respectively. In this method nanodiscs (ND) “which made up entirely of lipids extracted from cell cultures”2 have been employed as a carrier to solubilize the receptor (GSL) for numerous fundamental studies. Then the assay was prepared by mixing the glycosphingolipid-nanodisc complex with the solution containing soluble proteins. The resulting assay was transferred to the gas phase by ESI. The authors implied that the Direct ESI-MS of the protein-GSL-ND is not upfront. Thus, collision-induced dissociation (CID) method has been used to allow the detachment of the phospholipid bonds between protein-receptor and nanodisc. Following this cleavage, the remaining isolated protein-GSL complex bonds have been broken by another step of CID. Finally the mass analysis of generated fragments in conjunction with ion mobility separation (IMS) which is useful method when isomers of GSL are present employed to identify receptors. This method provides many useful outcomes. For example, they found no evidence of interactions between CT or Stx2 with their non-native GSL in their ESI mass spectra which reveals that proteins just have certain interactions with their native GSL.
Finally, they claimed that CaR-ESI-MS can be employed for detecting special protein-glycosphingolipid interactions and the possibility of using this strategy to discover unknown receptors from a complex mixture. However; there is still some work needed to be done for further discoveries about the application of this method to evaluate relative attractions for different GSL. As a complimentary I thought that electron spin resonance (ESR) and ultraviolet resonance Raman spectroscopy (UVRR) might be useful to investigate the protein-receptor bindings and the selectivity of the interactions between proteins and lipids.

Reference:
1, 2- These are come directly from this paper.

Title: Molecular Insight into Pt-Catalyzed Chemoselective Hydrogenation of an Aromatic Ketone by In Situ Modulation−Excitation IR Spectroscopy

By: Mengmeng Chen et al.

Journal: ACS Catalysis

The selective hydrogenation of aromatic ketones over supported noble metal catalysts is one of the key synthesis steps for the pharmaceutical industry. The mechanisms of these reactions, however, are not fully understood. To better understand the mechanisms for this class of reactions, the authors of this work used Modulation- Excitation and Attenuated Total Reflectance Infrared Spectroscopy (ATR-IR) to identify and monitor the kinetic behavior of the relevant surface species for the liquid phase selective hydrogenation of Acetophenone (AP) into 1-penylethanol (PE) over a gamma alumina (Al2O3) supported Pt catalyst.

The authors designed an ATR-IR cell in which a liquid stream of n-hexane and a stream of n-hexane mixed with AP could be alternatively pumped over the catalyst and the transient behavior of the surface species during reaction could be analyzed. It was discovered that, as the hydrogenation of AP into PE proceeded, a band associated with PE absorbed onto the surface of the Al2O3 support (1081 cm-1) appeared at the cost of the band associated with AP absorbed onto the Al2O3 surface (1672 cm-1), while the band associated with AP absorbed (1697 cm-1) onto the surface of the Pt catalyst appeared to reach a stable maximum. Furthermore, products associated with subsequent hydrogenation of PE were not observed in significant amounts during these experiments. Based upon these observations, the authors concluded that, in the liquid phase, the selective formation of PE over the products of further hydrogenation are a result of the tendency of PE to absorb more strongly onto the Al2O3 support than AP, thus preventing PE form absorbing and reacting on the Pt catalyst. The authors supported this conclusion by co-feeding AP and PE in the absence of hydrogen; finding that PE preferentially absorbed onto the Al2O3 support.

While the authors show that PE is able to displace absorbed AP on the surface of the Al2O3 support, they have not verified that the product’s affinity to the support is the reason behind the lack of subsequent reaction and high selectivity of PE. Another possibility may be that AP absorbs much more strongly to the platinum catalyst then PE and, thus, even without the favored sites for absorption on the support, the PE will not be able to occupy and react on the active sites of the Pt surface while the catalyst surface is saturated with absorbed AP species. This distinction could be resolved by comparing the dynamic in-situ ATR-IR spectra of AP hydrogenation between a normal catalyst sample and one in which the absorption sites on the Al2O3 support have been titrated with a basic probe molecule.

Minute Paper #1 (9/7/12) – Matt Irwin

Title: Bilayer Membrane Permeability of Ionic Liquid-Filled Block Copolymer Vesicles in Aqueous Solution
Authors: Z. Bai et al.
Journal: Macromolecules

In this article, the authors describe a new method for determining the permeability of the polymeric bilayer membrane of ionic liquid-filled vesicles dispersed in an aqueous solution. Their method involves using fluorescent spectroscopy to monitor the rate at which a fluorescent dye in the vesicles is extinguished by a quencher molecule that must diffuse across the vesicle membrane. Analytical techniques used in this study include laser scanning confocal microscopy, fluorescence spectroscopy, ultraviolet-visible spectroscopy, proton nuclear magnetic resonance spectroscopy, and dynamic light scattering.

The vesicles in the study were formed via the self-assembly of poly((1,2-butadiene)-b-(ethylene oxide)) (PB-b-PEO) in solution in which the PB block forms the membrane and the PEO block forms the corona. Two different size block copolymers were used in this study: PB-PEO(14-4.5) and PB-PEO(6.7-2.4), where the numbers in parentheses represents the block molecular weights in kg/mol. These block copolymers yielded membrane thicknesses of approximately 28 nm an 18 nm, respectively. The vesicles were charged with the ionic liquid 1-ethyl-3-methyl-imidazolium bis(trifluoromethylsulfonyl)amide ([EMIM][TFSA]) and the fluorescent dye Nile Red, and the quencher molecule dichloroacetamide was added to the aqueous phase. The permeability of the liquid PB membrane was then determined by monitoring the rate at which the fluorescence of the vesicles in the aqueous phase diminished as a function of time. Specifically, fluorescence spectroscopy measurements were performed using a HeNe laser with an excitation wavelength of 543 nm to excite the dye, and the fluorescence of the solution was measured over 635 nm to 645 nm as a function of time. The authors found that the membrane permeability was quite high due to the softness of the liquid PB membrane. In addition, decreasing the membrane thickness from 28 nm to 18 nm resulted in a remarkable 5-fold increase in membrane permeability.

Despite the exciting results in this paper, the authors leave many questions unanswered. In the introduction, the authors note that these ionic liquid-filled vesicle systems could be used as nanoreactors. Therefore, the ability to select a desired membrane permeability based on necessary reaction conditions would be highly useful. In order to achieve this, a quantitative relationship between the membrane thickness and permeability could be developed by investigating additional block copolymer sizes. In addition, the authors of this paper only investigate one dye-quencher pair. In order to confirm the universality of the observed relationship between membrane thickness and permeability, additional studies with other dyes and quenchers should be performed.

Title: Fluorescence Imaging of Cellular Metabolites with RNA
By: SR Jaffrey et al.
Journal: Science

In this paper, the authors want to see real-time metabolism using biological sensor. They focus on RNA as sensor, not protein, because its easiness of synthesis in vitro. They synthesize RNA using PCR technique and they use HPLC, spectrophotometer, fluorescence spectrometer, and optical microscope with CCD camera.
They synthesize RNA sensor composed of Spinach, a transducer, and a target-binding aptamer. When aptamer attach to target molecule, transducer starts to change and cause folding of Spinach. Changed Spinach form fluorescent complex with the fluorophore 3,5-difluoro-4-hydroxybenzylidene imidazolinone(DFHBI). They thermodynamically design the sensor that emits fluorescence only when target molecule is attached to aptamer. They synthesize sensors for adenosine, ADP, SAM, guanine and GTP. They find increase of fluorescence is linear in physiological concentration ranges. And, of course, sensors are not related to metabolism. They monitor level of SAM according to concentration of methionine, the SAM precursor. Also, they find the role of S-adenosyl-homocysteine(SAH) hydrolase in SAM pathway, which recycle SAH, byproducts of SAM. They also monitor the level of ADP. They say RNA sensor is better than previous Förster resonance energy transfer (FRET) sensor because it gives brighter fluorescence.
In my opinion, they need to apply it to in vivo. They need to test whether this RNA sensor can selectively scan target molecule in vivo. For first step, they can measure it with several mixtures such as ADP, ATP, and GTP. They also can strengthen the specific selectivity of their sensor by measuring equilibrium constant of the sensor to target molecule. They need to show there is big difference in Kd between target molecule and similar molecules. They can synthesize other sensors detect more important molecules such as specific molecule of cancer cell.

Glen, leuk dat je er bent! Neem even de tijd en bekijk de forum regels. Ik denk dat ze zal helpen om comfortabel met hoe dingen werken hier in de buurt, evenals kennis te maken met de admins en mods.

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