Analytical Window Research Articles

Expansion of the Analytical Window for Oil Spill Characterization by Ultrahigh Resolution Mass Spectrometry: Beyond Gas Chromatography

Traditional tools for routine environmental analysis and forensic chemistry of petroleum have relied almost exclusively on gas chromatography-mass spectrometry (GC-MS), although many compounds in crude oil (and its transformation products) are not chromatographically separated or amenable to GC-MS due to volatility. To enhance current and future studies on the fate, transport, and fingerprinting of the Macondo well oil released from the 2010 Deepwater Horizon disaster, we created an extensive molecular library of the unadulterated petroleum to compare to a tar ball collected on the beach of Louisiana. We apply ultrahigh resolution Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry to identify compositional changes at the molecular level between native and weathered crude oil samples and reveal enrichment in polar compounds inaccessible by GC-based characterization. The outlined approach provides unprecedented detail with the potential to enhance insight into the environmental fate of spilled oil, improved toxicology, molecular modeling of biotic/abiotic weathering, and comprehensive molecular characterization for petroleum-derived releases. Here, we characterize more than 30,000 acidic, basic, and nonpolar unique neutral elemental compositions for the Macondo well crude oil, to provide an archive for future chemical analyses of the environmental consequences of the oil spill.

Requiem for a CAMP: The life and death of a domestic U.S. drug war institution

The life and death of California's Campaign Against Marijuana Planting (CAMP, 1983–2012) offers a unique analytical window into the time and space of the U.S. war on drugs in a global context. This paper draws on CAMP report archives, ethnographic interviews, and secondary data sources to locate the significance of CAMP, its demise, and enduring legacy for the political economy of domestic illicit cannabis production in southern Humboldt County, where it was initially focused. I first introduce the economic geography of cannabis production in southern Humboldt County and California. In the first part of the paper, using theoretical frameworks from Critical Geopolitics and International Relations, I examine the geo-politics of CAMP's emergence. In the second part of the paper, I examine industrial reterritorialization associated with its geographies of enforcement over time. I conclude by discussing the eclipse of its foundational logic-and-practice (policing the “Emerald Triangle”) by new political and economic geographies of power.

Synthesis and stability study of a new major metabolite of γ-hydroxybutyric acid.

γ-Hydroxybutanoic acid (GHB) is used as a date-rape drug, which renders the victims unconscious and defenceless. Intoxications are very difficult to detect for forensic scientists due to rapid metabolism to endogenous levels of GHB. We recently discovered a new major metabolite, 2, of GHB (1) that could potentially extend the analytical detection window for GHB intoxications. Herein we disclose synthetic procedures based on a Koenigs–Knorr glucuronidation approach that provides GHB glucuronide 2 and a deuterium-labelled analogue d4-2 of high purity suitable for analytical chemistry. In addition, we have assessed the stability of GHB glucuronide 2 by mimicking the natural pH range for urine, which is of importance in the development of new analytical methods. Using NMR we show that GHB glucuronide 2 is highly stable towards aqueous hydrolysis within the pH range normally observed for urine even at elevated temperature.

Measurement of iron chemical speciation in seawater at 4 °C: The use of competitive ligand exchange–adsorptive cathodic stripping voltammetry

Iron is mostly bound to poorly characterised organic ligands; thus, organic ligands are paramount in defining Fe biogeochemical cycling and its control on oceanic primary productivity. Since 1994, Fe chemical speciation has been determined by Competitive Ligand Exchange–Adsorptive Cathodic Stripping Voltammetry (CLE–AdCSV) at room temperature. However, chemical speciation is strongly dependent on temperature and some organic ligands can be temperature sensitive. Here, we compare the use of the CLE–AdCSV at room temperature and at 4°C—a temperature closer to that found in the Southern Ocean, one of the largest iron-limited regions. For both temperatures, similar detection limits and total Fe concentrations were found. However, at 4°C the analytical detection window (αFe(TAC)2) was shifted by 1.4-fold towards the detection of weaker ligands, resulting in up to 2-fold lower ligand concentrations as well as a 2- to 5-fold and 10- to70-fold lower conditional stability constants with inorganic Fe (Fe′) and Fe(III), respectively. As a result, the Fe′ concentration at 4°C was 2-fold greater, resulting in direct implication for Fe bioavailability. Results show that difference in Fe chemical speciation at 4°C was not solely explained by temperature effect on thermodynamics with the exchange ligands or the diffusion of the electroactive complex towards the Hg drop. Lowering analytical window during analysis at room temperature is proposed as a first estimate of temperature effect on iron chemical speciation.

Preface "Sources and rapid biogeochemical transformation of dissolved organic matter in the Atlantic surface ocean"

One of the most important issues needed to be studied to better understand global fluxes of marine organic matter is to resolve its molecular characteristics and mechanisms which convert fresh, labile biomolecules into semi-labile and refractory dissolved organic compounds (Jiao et al., 2010) which resist degradation for an average time of 5000 years (Bauer et al., 1992; Williams and Druffel, 1987). From a chemical point of view, the persistence of marine dissolved organic matter (DOM) is very unusual. The mostly oxygenand nutrient-rich oceanic water column should be conducive to a rapid microbial degradation of organic matter and subsequent release of CO2. Contrary to this expectation, however, a significant portion of the atmospheric carbon remains in the DOM of the oceans and circulates in global currents on long time scales. Despite the importance of gaining better insights into the molecular mechanisms affecting organic carbon dynamics, very little is known about these processes. Thus, recent reports from the intergovernmental panel on climate change (Denman et al., 2007) barely considered potential changes in quality, quantity and fluxes of marine organic matter. In the oceans, dissolved organic substances constitute an important source of organic carbon and organic nitrogen (Hansell et al., 2009). The pool of dissolved organic carbon (DOC) exceeds the amount of carbon stored in marine animals, plants and bacteria by a factor of about 200. The majority of DOM in the oceans is originally formed from CO2 by plankton and land plants during photosynthesis. The DOM, resulting from this organic material, is directly released into the water by marine plankton and during the degradation of these organisms. Also, DOM can be transported into the oceans via rivers and atmospheric deposits via aerosols. A small fraction of marine DOM, such as persistent organic pollutants or black carbon derivatives, can be derived from anthropogenic activities. DOM can bind to trace metals such as iron and is therefore also responsible for their distribution in the sea. The complex composition of DOM is an enormous analytical challenge, resulting in sparse information on chemical structures. The DOM complexity prevents resolution of single molecules by chromatographic separation or conventional mass spectrometry. However, within the last 10 years analytical instruments, which can deliver molecular signals, improved tremendously. Namely, improvements of nuclear magnetic resonance spectroscopy (NMR) and Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) opened a new analytical window for the molecular characterization of natural organic matter (e.g. Stenson et al., 2003; Kujawinski et al., 2004; Koch et al., 2005; Hertkorn et al., 2008; Witt et al., 2009). Meridional transits of polar research vessels offer a great opportunity to perform long-distance surface water transects with continuous water and air sampling, since frequent stops for long station work are usually not possible during such transit cruises. Trace metal (Pohl et al., 2011; Helmers, 1996) and plankton studies (Robinson et al., 2009) and a recent GEOTRACES pilot study in the Atlantic Ocean (Rutgers van der Loeff, 2007) are great examples for the importance of such projects. Previous work along the north–south transect also provided a high resolution comparison of bacterial and primary production (Hoppe et al., 2002). The authors suggested that the observed net heterotrophy required

Insulin increases central apolipoprotein E levels as revealed by an improved technique for collection of cerebrospinal fluid from rats

Cerebrospinal fluid (CSF) provides an invaluable analytical window to the central nervous system (CNS) because it reflects the dynamically changing complement of CNS constituents. We describe an improved method for sampling CSF in rats that is easy to perform. It has a 96% success rate of CSF collection and consistently yields large volumes (150–200μl) of CSF. The blood contamination rate is also low (6%) as determined by both visual inspection and the lack of molecular detection of apolipoprotein B, a plasma-derived protein, which is absent in the CNS. This improved method of CSF sampling can have broad applicability in physiological and pharmacological evaluation for diverse CNS targets. We used this technique to provide proof of principle by examining the effect of intraperitoneal insulin on the level of apolipoprotein E (apoE) in the CSF. Insulin (0.5 and 1U/kg) led to a significant increase of insulin in both plasma and CSF at 2h after intraperitoneal administration and decreased blood glucose for at least 2h. ApoE concentrations in CSF, but not in plasma, were also significantly increased, and its time-course was inversely correlated with the alterations in blood glucose over 2h. These results provide a pharmacological validation of the novel CSF sampling and validation procedure for sampling rat CSF.

Analytical Challenges in Environmental and Geosciences

New emerging issues in environmental science can often be linked to the development of new analytical techniques and methods. A prominent example is the introduction of the electron capture detector in the 1960s. The electron capture detector is a highly selective and sensitive detector for halogenated compounds and opened an analytical window for the investigation of the ubiquitous occurrence of halogenated insecticides such as DDT and polychlorinated biphenyls in environmental matrices and animal tissues. In the last 15 years the rapid development of liquid chromatography–mass spectrometry (LC-MS) instruments with increasing sensitivity and selectivity has opened another analytical window for polar, not well degradable water pollutants. Among these so-called new emerging pollutants are pharmaceuticals and personal care products (PhPCPs), a very large and diverse group of chemicals occurring in the water cycle. A recent publication of the German Federal Environment Agency (Umweltbundesamt) reports on monitoring data for more than 270 pharmaceuticals and their metabolites in the literature. The challenge in the analysis of PhPCPs and other new emerging compounds is the selection of the most relevant compounds from a huge list of chemicals in daily use. In Europe this list comprises several tens of thousands of compounds from a total of more than 120,000 registered chemicals. The consideration of metabolites and abiotic transformation products of the original pollutants in surface water and groundwater and in wastewater and drinking water treatment further increases the number of relevant compounds and requires detailed knowledge of chemical and biological transformation processes. In environmental science the general questions for analytical chemists are still the same as 25 years ago during my university education: what compounds occurs in what quantity, where, and why? Preferably, sensitive and selective analytical methods are the basis to answer these questions. Most important today, especially for organic pollutants, are separation techniques such as gas chromatography–mass spectrometry and LC-MS. Other approaches include classic and miniaturized applications of spectroscopy, electrochemistry, or molecular recognition by antibodies or specific natural or synthetic receptors. The latter are also important in effect-directed analysis, which aims to link biological effects to compound identification and quantitation. Immunoassays such as enzyme-linked immunosorbent assays are very useful tools for rapid, inexpensive, and portable detection of specific analytes. The challenge is still the generation of specific antibodies for a broader range of relevant compounds. Multicompound detection and identification is the domain of nontarget approaches by high-resolution mass spectrometry, which is based on the determination of the molecular formulae and has experienced increasing application owing to recent developments of sensitive and reliable quadrupole time-of-flight and Orbitrap instruments. The main challenges are the selection of relevant signals from the large data sets and the elucidation of the compound structures of unknowns. This requires statistical approaches to select compounds of interest and further structural information from mass fragmentation and often nuclear magnetic resonance (NMR) spectroscopy to elucidate structural isomers. Despite increased NMR sensitivity, there is still a Published in the topical collection Analytical Challenges in Environmental and Geosciences with guest editor Christian Zwiener.

Molecularly imprinted polymers as optical sensing receptors: Correlation between analytical signals and binding isotherms

Despite the increasing number of usage of molecularly imprinted polymers (MIPs) in optical sensor application, the correlation between the analytical signals and the binding isotherms has yet to be fully understood. This work investigates the relationship between the signals generated from MIPs sensors to its respective binding affinity variables generated using binding isotherm models. Two different systems based on the imprinting of metal ion and organic compound have been selected for the study, which employed reflectance and fluorescence sensing schemes, respectively. Batch binding analysis using the standard binding isotherm models was employed to evaluate the affinity of the binding sites. Evaluation using the discrete bi-Langmuir isotherm model found both the MIPs studied have generally two classes of binding sites that was of low and high affinities, while the continuous Freundlich isotherm model has successfully generated a distribution of affinities within the investigated analytical window. When the MIPs were incorporated as sensing receptors, the changes in the analytical signal due to different analyte concentrations were found to have direct correlation with the binding isotherm variables. Further data analyses based on this observation have generated robust models representing the analytical performance of the optical sensors. The best constructed model describing the sensing trend for each of the sensor has been tested and demonstrated to give accurate prediction of concentration for a series of spiked analytes.

Early-Elimination Modification for Priority-First Search Decoding

In order to release the growing demand for computational complexity with respect to increasing information sequence length in the priority-first search decoding algorithm, a path elimination modification is proposed and also analyzed in this work. Specifically, we propose to directly eliminate all paths whose end nodes are Δ-level prior to the farthest node among those that have been visited thus far by the priority-first search. Following the argument on random coding, we then analyze the path elimination window Δ that results in a larger exponent for additional decoding error caused by path elimination than the exponent of the maximum-likelihood error performance, and hence guarantees exponentially negligible performance degradation. Our analytical results indicate that under additive white Gaussian noise (AWGN) channels, the path elimination window required for exponentially negligible performance degradation is just three times the code constraint length for rate one-half convolutional codes. It can be further reduced to 1.7-fold of the code constraint length when rate one-third convolutional codes are considered instead. Simulation results confirm these analytical window sizes. As a consequence, the priority-first search decoding algorithm can considerably reduce its computation burden and memory consumption by directly eliminating a large number of paths with nearly no performance degradation. This makes the priority-first search decoding algorithm with path elimination suitable for applications that demand low-complexity software implementation with near optimal performance.

Intramolecular stable carbon isotopic analysis of archaeal glycosyl tetraether lipids

Glycolipids are prominent constituents in the membranes of cells from all domains of life. For example, diglycosyl-glycerol dibiphytanyl glycerol tetraethers (2Gly-GDGTs) are associated with methanotrophic ANME-1 archaea and heterotrophic benthic archaea, two archaeal groups of global biogeochemical importance. The hydrophobic biphytane moieties of 2Gly-GDGTs from these two uncultivated archaeal groups exhibit distinct carbon isotopic compositions. To explore whether the isotopic compositions of the sugar headgroups provide additional information on the metabolism of their producers, we developed a procedure to analyze the δ(13)C values of glycosidic headgroups. Successful determination was achieved by (1) monitoring the contamination from free sugars during lipid extraction and preparation, (2) optimizing the hydrolytic conditions for glycolipids, and (3) derivatizing the resulting sugars into aldononitrile acetate derivatives, which are stable enough to withstand a subsequent column purification step. First results of δ(13)C values of sugars cleaved from 2Gly-GDGTs in two marine sediment samples, one containing predominantly ANME-1 archaea and the other benthic archaea, were obtained and compared with the δ(13)C values of the corresponding biphytanes. In both samples the dominant sugar headgroups were enriched in (13)C relative to the corresponding major biphytane. This (13)C enrichment was significantly larger in the putative major glycolipids from ANME-1 archaea (∼15‰) than in those from benthic archaea (<7‰). This method opens a new analytical window for the examination of carbon isotopic relationships between sugars and lipids in uncultivated organisms.

The Use of Surface-Enhanced Raman Scattering for Detecting Molecular Evidence of Life in Rocks, Sediments, and Sedimentary Deposits

Raman spectroscopy is a versatile analytical technique capable of characterizing the composition of both inorganic and organic materials. Consequently, it is frequently suggested as a payload on many planetary landers. Only approximately 1 in every 10(6) photons are Raman scattered; therefore, the detection of trace quantities of an analyte dispersed in a sample matrix can be much harder to achieve. To overcome this, surface-enhanced Raman scattering (SERS) and surface-enhanced resonance Raman scattering (SERRS) both provide greatly enhanced signals (enhancements between 10(5) and 10(9)) through the analyte's interaction with the locally generated surface plasmons, which occur at a "roughened" or nanostructured metallic surface (e.g., Cu, Au, and Ag). Both SERS and SERRS may therefore provide a viable technique for trace analysis of samples. In this paper, we describe the development of SERS assays for analyzing trace amounts of compounds present in the solvent extracts of sedimentary deposits. These assays were used to detect biological pigments present in an Arctic microoasis (a small locale of elevated biological productivity) and its detrital regolith, characterize the pigmentation of microbial mats around hydrothermal springs, and detect fossil organic matter in hydrothermal deposits. These field study examples demonstrate that SERS technology is sufficiently mature to be applied to many astrobiological analog studies on Earth. Many current and proposed imaging systems intended for remote deployment already posses the instrumental components needed for SERS. The addition of wet chemistry sample processing facilities to these instruments could yield field-deployable analytical instruments with a broadened analytical window for detecting organic compounds with a biological or geological origin.

Differential scanning calorimetry of blood plasma for lung cancer diagnosis.

e21085 Background: We have invented a novel assay by using differential scanning calorimetry (DSC) for rapid characterization of the plasma proteome based on the thermal properties of the constituent proteins. Human plasma is a complex fluid that contains more than 3000 individual proteins and peptides in different quantities. DSC is a proven biophysical technique for measuring the energy absorbed by a solution of proteins as a function of temperature. The assay offers an entirely new analytical window into the plasma proteome, and provides a signature calorimetric thermogram that is a unique snapshot of an individual's plasma protein composition. Methods: Plasma samples were collected from lung cancer (LC) patients and healthy volunteers and stored at –70°C. Sample analysis was performed using an automated capillary DSC. Comparison of plasma thermograms from LC patients and controls was done using quantile-quantile plots and the Kolmogorov-Smirnov test. Results: Since our pilot study in 2009, we enrolled 60 LC patients but only analyzed 30 samples for this validation study plus another 109 normal controls. The thermograms from 109 control plasmas were highly reproducible and showed similar characteristic shape without statistical difference from our previous 100 control results. The samples from 30 new LC patients also yielded a unique thermogram differing from that of current and previous controls but similar to the results from 20 previous LC patients in our pilot study in 2009 without statistical difference. Our results further validated our hypothesis that DSC was sensitive to properties of the plasma proteins other than their charge and mass, and changes in thermogram shapes and positions in LC may arise from changes in the concentrations of plasma proteins, from disease-induced protein-protein interactions, or from the secretion into plasma of disease-related peptides or ligands that may bind to the most abundant plasma proteins. Conclusions: Lung cancer may have a characteristic signature thermogram that may serve as a biomarker. Author Disclosure Employment or Leadership Position Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Louisville Biosciences, Inc.

Fluorescent duplex allele-specific PCR and amplicon melting for rapid homogeneous mtDNA haplogroup H screening and sensitive mixture detection.

BackgroundFor large scale studies aiming at a better understanding of mitochondrial DNA (mtDNA), sequence variation in particular mt haplogroups (hgs) and population structure, reliable low-cost high-throughput genotyping assays are needed. Furthermore, methods facilitating sensitive mixture detection and relative quantification of allele proportions are indispensable for the study of heteroplasmy, mitochondrial sequence evolution, and mitochondrial disorders. Here the properties of a homogeneous competitive duplex allele specific PCR (ARMS) assay were scrutinized in the light of these requirements.Methodology/Principal FindingsA duplex ARMS assay amplifying either the ancestral mtDNA 2706G allele (non-hg H samples) or the derived 7028C allele (hg H samples) in the presence of SYBR Green fluorescent reporter dye was developed and characterized. Product detection, allele calling, and hg inference were based on the amplicon-characteristic melting-point temperatures obtained with on-line post-PCR fluorescent dissociation curve analysis (DCA). The analytical window of the assay covered at least 5 orders of magnitude of template DNA input with a detection limit in the low picogram range of genomic DNA. A set of forensically relevant test specimens was analyzed successfully. The presence of mtDNA mixtures was detected over a broad range of input DNA amounts and mixture ratios, and the estimation of allele proportions in samples with known total mtDNA content was feasible with limitations. A qualified DNA analyst successfully analyzed ∼2,200 DNA extracts within three regular working days, without using robotic lab-equipment. By performing the amplification on-line, the assay also facilitated absolute mtDNA quantification.ConclusionsAlthough this assay was developed just for a particular purpose, the approach is general in that it is potentially suitable in a broad variety of assay-layouts for many other applications, including the analysis of mixtures. Homogeneous ARMS-DCA is a valuable tool for large-volume studies targeting small numbers of single nucleotide polymorphisms (SNPs).

(Mis)adventures in Rumsfeld space

Ewald’s recent genealogy of constructions of risk in Western societies argues that the 1980s saw an important paradigm shift to the “precautionary principle”. Critical scholars have taken up this idea as a lens through which to interpret the Bush administration’s ‘war on terror’. I argue that 11 September 2001 actually brought about qualitative changes to this paradigm. Bush’s pre-emptive doctrine is driven, and perhaps even more importantly, continually justified to the US population, by what might be called the “trans-precautionary principle”, a move from “decisionism” to “actionism”, and a new radicalization of the way fear is produced and managed. Donald Rumsfeld’s famous typology of different articulations of knowledge and ignorance offers an excellent analytical window onto the connections between ignorance, fear and geopolitical action in this new regime. In the latter part of the essay, his four modes of knowledge/ignorance are arrayed, for heuristic purposes, in an abstract spatial grid organized along dimensions of the specificity and possession of knowledge. This allows a ‘mapping’ of some of the Bush administration’s more controversial strategies in the ‘war on terror’, as a set of different pathways through Rumsfeld Space.

"Come Back Home": Regional Travels, Global Encounters, and Local Nostalgias in Bahamian Popular Musics

Bahamian musical life has been deeply informed and shaped by at least three separate but interrelated themes: 1) the physical interposition of the Bahamas between the United States and the rest of the Caribbean; 2) tourism: and 3) the nation's colonial and postcolonial histories. These geographic, economic, and political influences are unthinkable without considering the ways that travel is implicated in each of them. Travel operates at several registers in the Bahamas, including at least physical travels, musical migrations and media flows, mobilities informed by center/periphery dynamics, and journeys related to time and nostalgia. Rake-n-scrape music opens an analytical window onto these spatial, economic, and socio-political registers within the Bahamas. This article thus traces the rake-n-scrape's movement from peripheral, traditional music to popular, urban soundtrack and follows this with an exploration of the politics attendant to the genre's more recent repositioning as roots music.

Copper complexation in coastal rainwater, southeastern USA

Complexation of dissolved copper (Cu) was studied in Atlantic coastal rainwater using adsorptive cathodic stripping voltammetry (ASCV) with salicylaldoxime as a competing ligand at pH 7.8. Detectable concentrations of strong Cu-complexing ligands possessing conditional stability constants of 10 13–10 16 were observed in over 80% of the rain events occurring over the course of 2 year. In 11 of the 23 samples analysed, total dissolved Cu concentrations were higher than those of dissolved ligands, indicating that a significant fraction of the Cu occurred as the free ion and as weaker complexes. In the remaining samples, ligand concentrations were equal to or greater than Cu concentrations, indicating virtually complete (>99%) complexation of the ambient Cu. By varying the analytical detection window, two classes of ligands with differing conditional stability constants were detected in selected rain samples suggesting that the Cu ligands most likely represent a spectrum of organic compounds. Back trajectory analysis indicated that continentally dominated rain samples contained higher concentrations of Cu and organic ligands relative to storms of marine origin, suggesting a strong terrestrial and/or anthropogenic source of both Cu and ligands in rain at this location. Variability in Cu speciation may impact a variety of atmospheric redox reactions because free and complexed forms of the metal have very different reactivities.

Simultaneous determination of sildenafil and N-desmethyl sildenafil in human plasma by high-performance liquid chromatography method using electrochemical detection with application to a pharmacokinetic study

A method which employed high-performance liquid chromatography coupled with electrochemical detection was developed for the simultaneous determination of sildenafil and its metabolite, N-desmethyl sildenafil, in human plasma has. The method was developed and validated for purposes of its application to a pharmacokinetic study in healthy volunteers after an oral dose of 50 mg/tablet under fasting conditions. High precision and accuracy were demonstrated. A one-step liquid–liquid extraction further provides a simple and practical way to process plasma samples containing sildenafil with good quantitative recovery. Sampling lasted for 24 h after dosing; consequently a limit of quantitation (LOQ) of 7.858 ng/mL was achieved for sildenafil whereas a LOQ of 8.675 ng/mL was obtained for N-desmethyl sildenafil. The mobile phase consisted of acetonitrile, methanol and phosphate buffer (0.05 M) (18.5:34.5:47.0, v/v/v) pH 7.68. The stationary phase was a C 8 (150 mm × 4.6 mm), 5 μm particle size operated at 27 °C. All analytes were stable at the pH of the supernatant, and during the analytical time window. At the applied potential of +1.20 V versus Ag/AgCl, no interferences from endogenous plasma compounds were recorded at the retention times of sildenafil, N-desmethyl sildenafil. High resolution was obtained between the analytes and the employed internal standards.

Towards a comprehensive understanding of Bacillus subtilis cell physiology by physiological proteomics

Using Bacillus subtilis as a model system for functional genomics, this review will provide insights how proteomics can be used to bring the virtual life of genes to the real life of proteins. Physiological proteomics will generate a new and broad understanding of cellular physiology because the majority of proteins synthesized in the cell can be visualized. From a physiological point of view two major proteome fractions can be distinguished: proteomes of growing cells and proteomes of nongrowing cells. In the main analytical window almost 50% of the vegetative proteome expressed in growing cells of B. subtilis were identified. This proteomic view of growing cells can be employed for analyzing the regulation of entire metabolic pathways and thus opens the chance for a comprehensive understanding of metabolism and growth processes of bacteria. Proteomics, on the other hand, is also a useful tool for analyzing the adaptational network of nongrowing cells that consists of several partially overlapping regulation groups induced by stress/starvation stimuli. Furthermore, proteomic signatures for environmental stimuli can not only be applied to predict the physiological state of cells, but also offer various industrial applications from fermentation monitoring up to the analysis of the mode of action of drugs. Even if DNA array technologies currently provide a better overview of the gene expression profile than proteome approaches, the latter address biological problems in which they can not be replaced by mRNA profiling procedures. This proteomics of the second generation is a powerful tool for analyzing global control of protein stability, the protein interaction network, protein secretion or post-translational modifications of proteins on the way towards the elucidation of the mystery of life.

Two-dimensional reference map of Agrobacterium tumefaciens proteins.

Proteomics based on two-dimensional (2-D) gel electrophoresis of proteins followed by spot identification with mass spectrometry is a commonly used method for physiological studies. Physiological proteomics requires 2-D reference maps, on which most of the main proteins are identified. We present a reference map for the bacterial plant pathogen Agrobacterium tumefaciens proteins, which contains more than 300 entries with an isoelectric point (pI) between 4 and 7. The quantitative study of the proteins in the analytical window of the master gel demonstrated unique features, in comparison with other bacteria. In addition, a theoretical analysis of several protein parameters was performed and compared with the experimental results. A comparison of the theoretical molecular weight (MW) of the proteins and their theoretical pI with their vertical and horizontal migration distances, respectively, pointed out the existence of several proteins that strongly diverted from the graph trend-line. These proteins were clearly subjected to post-translational modifications, which changed their pI and/or MW. Additional support for post-translational modifications comes from the identification of multiple spots of the same gene products. Post-translational modifications appear to be more common than expected, at least for soluble proteins, as more than 10% of the proteins were associated with multiple spots.

Characterization of the imprint effect and the influence of imprinting conditions on affinity, capacity, and heterogeneity in molecularly imprinted polymers using the Freundlich isotherm-affinity distribution analysis.

Molecularly imprinted polymers (MIPs) have been used in a wide range of analytical applications in particular in chromatography and sensing. However, the binding properties in MIPs are typically measured only in a narrow concentration range, which corresponds to only a subset of the sites in MIPs. This limited analytical window and binding site heterogeneity of MIPs leads to inaccuracies and inconsistencies in the estimation of their binding properties. This has hampered the characterization and optimization of MIPs for analytical applications. In this study, the origins of the molecular imprinting effect were studied using the newly developed Freundlich isotherm-affinity distribution (FIAD) analysis. The analysis is able to readily calculate an affinity distribution for MIPs from the limited analytical window. The FIAD analysis also yields an estimate of number, affinity, and heterogeneity for this subset of binding sites. Consistent with previous studies, MIPs were found to have higher capacities than the corresponding nonimprinted polymers (NIPs). Interestingly, MIPs were also found to be more heterogeneous than NIPs. Examination of variables in the imprinting process including temperature, template concentration, and cross-linking percentages further confirmed these trends. Based on these observations, a model for the imprinting effect was developed. The larger population of high-affinity sites in MIPs appears to arise from a broadening of the heterogeneous distribution. This suggests that noncovalent MIPs may be ill-suited for chromatographic applications and other applications that are detrimentally affected by binding site heterogeneity and better suited to applications that are less affected by heterogeneity such as sensing.