Serum protein profiling by solid phase extraction and mass spectrometry: A future diagnostics tool?

Serum protein profiling by MS is a promising method for early detection of disease. Important characteristics for serum protein profiling are preanalytical factors, analytical reproducibility and high throughput. Problems related to preanalytical factors can be overcome by using standardized and rigorous sample collection and sample handling protocols. The sensitivity of the MS analysis relies on the quality of the sample; consequently, the blood sample preparation step is crucial to obtain pure and concentrated samples and enrichment of the proteins and peptides of interest. This review focuses on the serum sample preparation step prior to protein profiling by MALDI MS analysis, with particular focus on various SPE methods. The application of SPE techniques with different chromatographic properties such as RP, ion exchange, or affinity binding to isolate specific subsets of molecules (subproteomes) is advantageous for increasing resolution and sensitivity in the subsequent MS analysis. In addition, several of the SPE sample preparation methods are simple and scalable and have proven easy to automate for higher reproducibility and throughput, which is important in a clinical proteomics setting.     

Clinical application of breath biomarkers of oxidative stress status

Isolation and quantification of volatile breath biomarkers indicative of relevant alterations in clinical status has required development of new techniques and applications of existing analytical chemical methods. The most significant obstacles to successful application of this type of sample have been reduction in required sample volume permitting replicate analysis (an absolute requirement for all clinical studies), separation of the analyte(s) of interest from background molecules, water vapor and other molecules with similar physical properties, introduction of automation in analysis and the use of selective detection systems (electron impact mass spectrometry, flame photometric, thermionic detectors), and automated sample collection from the human subject. Advances in adsorption technology and trace gas analysis have permitted rapid progress in this area of clinical chemistry.     

Modelling the interactions of protein side-chains

The study of small model molecules containing the relevant functional groups can help us to understand the interactions between side-chains in proteins.Ab initio quantum chemical techniques allow the interactions between the model molecules to be studied with much greater accuracy than is possible for an entire protein, where the use of simple empirical potentials is the norm. In particular, the use ofab initio methods on model molecules permits us to incorporate the atom-atom anisotropic directionality of these interactions. We survey various methods of obtaining the components of theab initio interaction energy. These are then applied to three systems of biological interest. The first of these is the arginine/aspartate pair found in salt bridges, which involves hydrogen bonding between two charged species. Secondly, we look at the arginine/phosphotyrosine interaction found in complexes between SH2 domains and peptide ligands: here we find that the arginine/phosphate part of the interaction is energetically far more important than the arginine/aromatic part. Finally, we describe a detailed study of amino/aromatic interactions in proteins: unconventional hydrogen bonds are found to be remarkably uncommon relative to stacked geometries, and the reasons for this are examined.     

Measuring Diffusion Coefficients via Two-photon Fluorescence Recovery After Photobleaching

Multi-fluorescence recovery after photobleaching is a microscopy technique used to measure the diffusion coefficient (or analogous transport parameters) of macromolecules, and can be applied to both in vitro and in vivo biological systems. Multi-fluorescence recovery after photobleaching is performed by photobleaching a region of interest within a fluorescent sample using an intense laser flash, then attenuating the beam and monitoring the fluorescence as still-fluorescent molecules from outside the region of interest diffuse in to replace the photobleached molecules. We will begin our demonstration by aligning the laser beam through the Pockels Cell (laser modulator) and along the optical path through the laser scan box and objective lens to the sample. For simplicity, we will use a sample of aqueous fluorescent dye. We will then determine the proper experimental parameters for our sample including, monitor and bleaching powers, bleach duration, bin widths (for photon counting), and fluorescence recovery time. Next, we will describe the procedure for taking recovery curves, a process that can be largely automated via LabVIEW (National Instruments, Austin, TX) for enhanced throughput. Finally, the diffusion coefficient is determined by fitting the recovery data to the appropriate mathematical model using a least-squares fitting algorithm, readily programmable using software such as MATLAB (The Mathworks, Natick, MA).Download video file.^{(77M, mp4)}     

A simple assay for detection of small-molecule redox activity

In addition to selecting molecules of pharmacological interest, high-throughput screening campaigns often generate hits of undesirable mechanism, which cannot be exploited for drug discovery as they lead to obvious problems of specificity and developability. Examples of undesirable mechanisms are target alkylation/acylation and compound aggregation. Both types of "promiscuous" mechanisms have been described in the literature, as have methods for their detection. In addition to these mechanisms, compounds can also inhibit by oxidizing susceptible enzyme targets, such as metalloenzymes and cysteine-using enzymes. However, this redox phenomenon has been documented infrequently, and an easy method for detecting this behavior is missing. In this article, the authors describe direct proof of small-molecule oxidation of a cysteine protease by liquid chromatography/tandem mass spectrometry, develop a simple assay to predict this oxidizing behavior by compounds, and show the utility of this assay by demonstrating its ability to distinguish nuisance redox compounds from well-behaved inhibitors in 3 historical GlaxoSmithKline drug discovery efforts.     

Simultaneous Multicolor Imaging of Biological Structures with Fluorescence Photoactivation Localization Microscopy

Localization-based super resolution microscopy can be applied to obtain a spatial map (image) of the distribution of individual fluorescently labeled single molecules within a sample with a spatial resolution of tens of nanometers. Using either photoactivatable (PAFP) or photoswitchable (PSFP) fluorescent proteins fused to proteins of interest, or organic dyes conjugated to antibodies or other molecules of interest, fluorescence photoactivation localization microscopy (FPALM) can simultaneously image multiple species of molecules within single cells. By using the following approach, populations of large numbers (thousands to hundreds of thousands) of individual molecules are imaged in single cells and localized with a precision of ~10-30 nm. Data obtained can be applied to understanding the nanoscale spatial distributions of multiple protein types within a cell. One primary advantage of this technique is the dramatic increase in spatial resolution: while diffraction limits resolution to ~200-250 nm in conventional light microscopy, FPALM can image length scales more than an order of magnitude smaller. As many biological hypotheses concern the spatial relationships among different biomolecules, the improved resolution of FPALM can provide insight into questions of cellular organization which have previously been inaccessible to conventional fluorescence microscopy. In addition to detailing the methods for sample preparation and data acquisition, we here describe the optical setup for FPALM. One additional consideration for researchers wishing to do super-resolution microscopy is cost: in-house setups are significantly cheaper than most commercially available imaging machines. Limitations of this technique include the need for optimizing the labeling of molecules of interest within cell samples, and the need for post-processing software to visualize results. We here describe the use of PAFP and PSFP expression to image two protein species in fixed cells. Extension of the technique to living cells is also described.     

A simple water sampler, and a chamber for in situ incubation of plankton samples at discrete depths

• 1 A simple water sampler and an incubation chamber are presented. The water sampler is simpler in construction than the Ruttner sampler and other comparable devices. In addition to enclosure of water at a discrete depth, the incubation chamber releases a radioisotope or other substance of experimental interest into the sample.
• 2 Tests of performance are presented, demonstrating efficiency in enclosure of water from discrete depths, and time course of mixing of a tracer into the sample after activation of the release mechanism.

     

NMR studies of electrostatic potential distribution around biologically important molecules.

A new experimental approach has been developed to study the distribution of local electrostatic potential around specific protons in biologically important molecules. The approach is the development of a method denoted as "spin label/spin probe," which was proposed by one of us (. Mol. Biol. 6:498-507). The proposed method is based upon the quantitative measurement of the contribution of differently charged nitroxide probes to the spin lattice relaxation rate (1/T1) of protons in the molecule of interest, followed by calculation of local electrostatic potential using the classical Debye equation. In parallel, the theoretical calculation of potential distribution with the use of the MacSpartan Plus 1.0 program has been performed. Application of the method to solutions of simple organic molecules (aliphatic and aromatic alcohols, aliphatic carboxylates (propionate anion), and protonated ethyl amine and imidazole) allowed us to estimate the effective potential around the molecules under investigation. These were found to be in good agreement with theoretically expected values. This technique was then applied to zwitterionic amino acids bearing neutral and charged side chains (glycine, lysine, histidine, and aspartic acid). The reliability of the general approach is proved by the data presented in this paper. Application of this new methodology can afford insight into the biochemical significance of electrostatic effects in biological systems.     

Three algorithms and SAS macros for estimating power and sample size for logistic models with one or more independent variables of interest in the presence of covariates

Background

Commonly when designing studies, researchers propose to measure several independent variables in a regression model, a subset of which are identified as the main variables of interest while the rest are retained in a model as covariates or confounders. Power for linear regression in this setting can be calculated using SAS PROC POWER. There exists a void in estimating power for the logistic regression models in the same setting.

Methods

Currently, an approach that calculates power for only one variable of interest in the presence of other covariates for logistic regression is in common use and works well for this special case. In this paper we propose three related algorithms along with corresponding SAS macros that extend power estimation for one or more primary variables of interest in the presence of some confounders.

Results

The three proposed empirical algorithms employ likelihood ratio test to provide a user with either a power estimate for a given sample size, a quick sample size estimate for a given power, and an approximate power curve for a range of sample sizes. A user can specify odds ratios for a combination of binary, uniform and standard normal independent variables of interest, and or remaining covariates/confounders in the model, along with a correlation between variables.

Conclusions

These user friendly algorithms and macro tools are a promising solution that can fill the void for estimation of power for logistic regression when multiple independent variables are of interest, in the presence of additional covariates in the model.

     

A new DNA combing method for biochemical analysis

A simple molecular combing method for analysis of biochemical reactions, called the moving droplet method, has been developed. In this method, small droplets containing DNA molecules run down a sloped glass substrate, and this creates a moving interface among the air, droplet, and substrate that stretches the DNA molecules. This method requires a much smaller volume of sample solution than other established combing methods, allowing wider application in various fields. Using this method, λDNA molecules were stretched and absorbed to a glass substrate, and single-molecule analysis of DNA synthesis by DNA polymerases was performed.     

Manipulation and extraction of genomic DNA from cell lysate by functionalized magnetic particles for lab on a chip applications

A novel approach for extracting living cells' genomic DNA materials utilizing functionalized magnetic particles (MPs) is reported in this investigation. This strategy is amenable to handle bio-samples in a miniaturized environment and it offers a possibility to separate and purify DNA from other cell lysate mixtures "on-chip", which is known to be a bottle-neck step in an integrated micro-total-analysis-system (muTAS). "Species-specific" genomic DNA of interest is captured by the MPs based on the hybridization interaction between the biotinylated probes modified MPs and a complementary region of the targeted genome. The genome DNA anchored on the particles can be separated from the rest of cellular mixtures by a simple buffer washing upon the exertion of external magnetic force. Surface modifications of MPs and hybridization conditions affecting the genome capturing efficiency are investigated. Extraction of genomic DNA from E. coli is demonstrated in a silicon/glass-based micro-reactor patterned with a platinum heater and sensors. On-chip extraction and manipulation of genomic DNAs illustrated in this study is a step forward toward a total integrated bioanalytical microsystem for crude cells/sample analysis.     

A simple and effective sample preparation method for atomic force microscopy visualization of individual DNA molecules in situ

A simple, controllable and effective sample preparation method was established for atomic force microscopy (AFM) imaging of individual DNA molecules in aqueous solution. Firstly, magnesium ion (Mg²⁺) at a concentration of 5.0–10.0 mM as a positively charged bridge was transferred onto mica to immobilize DNA molecules. Then Mg²⁺-modified mica was used to investigate DNA molecules in any buffer without magnesium ion by AFM. AFM images demonstrated that DNA molecules can be successfully observed in solution with good resolution, reproducibility, and stability. Further, this DNA sample preparation method makes AFM successful to investigate DNA molecular interaction in situ and DNA/chitosan complex in gene delivery.     

Parvovirus-like particles as vaccine vectors.

A wide array of systems have been developed to improve "classic" vaccines. The use of small polypeptides able to elicit potent antibody and cytotoxic responses seems to have enormous potential in the design of safer vaccines. While peptide coupling to large soluble proteins such as keyhole limpet hemocyanin is the current method of choice for eliciting antibody responses and insertion in live viruses for cytotoxic T-lymphocyte responses, alternative cheaper and/or safer methods will clearly be required in the future. Virus-like particles constitute very immunogenic molecules that allow for covalent coupling of the epitopes of interest in a simple way. In this article, we detail the methodology employed for the preparation of efficient virus vectors as delivery systems. We used parvovirus as the model for the design of new vaccine vectors. Recently parvovirus-like particles have been engineered to express foreign polypeptides in certain positions, resulting in the production of large quantities of highly immunogenic peptides, and to induce strong antibody, helper-T-cell, and cytotoxic T-lymphocyte responses. We discuss the different alternatives and the necessary steps to carry out this process, placing special emphasis on the flow of decisions that need to be made during the project.     

Insights from soft X-rays: the chlorine and sulfur sub-structures of a CK2α/DRB complex

The diffraction pattern of a protein crystal is normally a product of the interference of electromagnetic waves scattered by electrons of the crystalline sample. The diffraction pattern undergoes systematic changes in case additionally X-ray absorption occurs, meaning if the wavelength of the primary X-ray beam is relatively close to the absorption edge of selected elements of the sample. The resulting effects are summarized as "anomalous dispersion" and can be always observed with "soft" X-rays (wavelength around 2 A) since they match the absorption edges of sulfur and chlorine. A particularly useful application of this phenomenon is the experimental detection of the sub-structures of the anomalous scatterers in protein crystals. We demonstrate this here with a crystal of a C-terminally truncated variant of human CK2alpha to which two molecules of the inhibitor 5,6-dichloro-1-beta-D: -ribo-furanosyl-benzimidazole (DRB) are bound. The structure of this co-crystal has been solved recently. For this study we measured an additional diffraction data set at a wavelength of 2 A which showed strong anomalous dispersion effects. On the basis of these effects we detected all sulfur atoms of the protein, the two liganded DRB molecules and a total of 16 additional chloride ions some of them emerging at positions filled with water molecules in previous structure determinations. A number of chloride ions are bound to structural and functional important locations fitting to the constitutive activity and the acidophilic substrate specificity of the enzyme.     

Influence of physical and chemical properties of HTSXT-FTIR samples on the quality of prediction models developed to determine absolute concentrations of total proteins,carbohydrates and triglycerides: a preliminary study on the determination of their absolute concentrations in fresh microalgal biomass

Absolute concentrations of total macromolecules (triglycerides, proteins and carbohydrates) in microorganisms can be rapidly measured by FTIR spectroscopy, but caution is needed to avoid non-specific experimental bias. Here, we assess the limits within which this approach can be used on model solutions of macromolecules of interest. We used the Bruker HTSXT-FTIR system. Our results show that the solid deposits obtained after the sampling procedure present physical and chemical properties that influence the quality of the absolute concentration prediction models (univariate and multivariate). The accuracy of the models was degraded by a factor of 2 or 3 outside the recommended concentration interval of 0.5–35 µg spot^?1. Change occurred notably in the sample hydrogen bond network, which could, however, be controlled using an internal probe (pseudohalide anion). We also demonstrate that for aqueous solutions, accurate prediction of total carbohydrate quantities (in glucose equivalent) could not be made unless a constant amount of protein was added to the model solution (BSA). The results of the prediction model for more complex solutions, here with two components: glucose and BSA, were very encouraging, suggesting that this FTIR approach could be used as a rapid quantification method for mixtures of molecules of interest, provided the limits of use of the HTSXT-FTIR method are precisely known and respected. This last finding opens the way to direct quantification of total molecules of interest in more complex matrices.     

A simple molecular model for thermophilic adaptation of functional nucleic acids

RNA molecules have numerous functions including catalysis and small molecule recognition, which typically arise from a tertiary structure. There is increasing interest in mechanisms for the thermostability of functional RNA molecules. Sosnick, Pan, and co-workers introduced the notion of "functional stability" as the free energy of the tertiary (functional) state relative to the next most stable (nonfunctional) state. We investigated the extent to which secondary structure stability influences the functional stability of nucleic acids. Intramolecularly folding DNA triplexes containing alternating T*AT and C+*GC base triples were used as a three-state model for the folding of nucleic acids with functional tertiary structures. A four-base-pair tunable region was included adjacent to the triplex-forming portion of the helix to allow secondary structure strength to be modulated. The degree of folding cooperativity was controlled by pH, with high cooperativity maintained by lower pH (5.5), and no cooperativity by higher pH (7.0). We find a linear relationship between functional free energy and the free energy of the secondary structure element adjacent to tertiary interactions, but only when folding is cooperative. We translate the definition of functional stability into equations and perform simulations of the thermodynamic data, which lend support to this model. The ability to increase the melting temperature of tertiary structure by strengthening base-pairing interactions separate from tertiary interactions provides a simple means for evolving thermostability in functional RNAs.     

Recent advances in quantitative colocalization analysis: Focus on neuroscience

Quantitative colocalization analysis is an advanced digital imaging tool to characterize the spatial expression of molecules of interest in immunofluorescence images obtained using confocal microscopes. It began from simple pixel counting and, with introduction of specialized algorithms, transformed into a powerful image analyzing technique capable of identifying the exact locations of various molecules in tissues and cells and describing their subtle changes in dynamics. Applications of quantitative colocalization in the field of neuroscience proved to be particularly informative by helping to obtain observations not otherwise achievable using other techniques. In this article, we review the background and applicability of quantitative colocalization with special focus on neuroscience research.     

Aromatic and heteroatom-containing organic compounds in the lunar samples

The data concerning the indigenous organic compounds in the lunar samples has been consistent. The Apollo 11 sample appeared to be moderately contaminated, and most investigators found known terrestrial artifacts in these samples. The Apollo 12 data indicated that perhaps benzene and toluene were indigenous to the Moon at concentrations below 100 parts per billion. Other, more complex organic molecules (in particular of aromatic structure) might also be present, but in concentrations below 1 ppb. In general, the structural types reported have been relatively simple; perhaps indicating that what little organic chemistry occurs on the lunar surface can best be described as reactions between individual atoms of carbon, hydrogen, oxygen and nitrogen.     

Hydrodynamic guiding for addressing subsets of immobilized cells and molecules in microfluidic systems

Background

The interest in microfluidics and surface patterning is increasing as the use of these technologies in diverse biomedical applications is substantiated. Controlled molecular and cellular surface patterning is a costly and time-consuming process. Methods for keeping multiple separate experimental conditions on a patterned area are, therefore, needed to amplify the amount of biological information that can be retrieved from a patterned surface area. We describe, in three examples of biomedical applications, how this can be achieved in an open microfluidic system, by hydrodynamically guiding sample fluid over biological molecules and living cells immobilized on a surface.

Results

A microfluidic format of a standard assay for cell-membrane integrity showed a fast and dose-dependent toxicity of saponin on mammalian cells. A model of the interactions of human mononuclear leukocytes and endothelial cells was established. By contrast to static adhesion assays, cell-cell adhesion in this dynamic model depended on cytokine-mediated activation of both endothelial and blood cells. The microfluidic system allowed the use of unprocessed blood as sample material, and a specific and fast immunoassay for measuring the concentration of C-reactive protein in whole blood was demonstrated.

Conclusion

The use of hydrodynamic guiding made multiple and dynamic experimental conditions on a small surface area possible. The ability to change the direction of flow and produce two-dimensional grids can increase the number of reactions per surface area even further. The described microfluidic system is widely applicable, and can take advantage of surfaces produced by current and future techniques for patterning in the micro- and nanometer scale.