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Analytical and Bioanalytical Chemistry (v.389, #4)

Focus on proteomics by Wolf D. Lehmann (pp. 989-989).

is group leader at the Core Facility Molecular Structure Analysis of the German Cancer Research Center (DKFZ) in Heidelberg. His research interests are collision-induced fragmentation of modified peptides and analysis of protein phosphorylation by tandem mass spectrometry, liquid chromatography–tandem mass spectrometry and element mass spectrometry.

Sample preparation by in-gel digestion for mass spectrometry-based proteomics by Bernhard Granvogl; Matthias Plöscher; Lutz Andreas Eichacker (pp. 991-1002).

The proteomic characterization of proteins and protein complexes from cells and cell organelles is the next challenge for investigation of the cell. After isolation of the cell compartment, three steps have to be performed in the laboratory to yield information about the proteins present. The protein mixtures must be separated into single species, broken down into peptides, and, finally, identified by mass spectrometry. Most scientists engaged in proteomics separate proteins by electrophoresis. For characterization and identification of proteomes, mass spectrometry of peptides is the method of choice. To combine electrophoresis and mass spectrometry, sample preparation by “in-gel digestion” has been developed. Many procedures are available for in-gel digestion, which inspired us to review in-gel digestion approaches. Figure Classical in-gel digestion process for a protein band stained with CBB. Protein bands are cut from the polyacrylamide gel (1). CBB molecules (blue circles) bound to the protein are released by iterative incubation in a buffered organic solvent system (2). To increase digestion efficiency and sequence coverage proteins are reduced (3) and alkylated (4). Proteins are subsequently digested with proteolytic enzymes (scissors symbols), typically trypsin (5). Trypsin cleaves at the amino acid residues arginine (R) and lysine (K). The resulting peptides (A, B, and C) are extracted from the polyacrylamide matrix (6). The peptide solution can be further purified for analysis by mass spectrometry (Section “Concentration and desalting of peptides”)

Keywords: In-gel digestion; Trypsin; Mass spectrometry

Collision-induced reporter fragmentations for identification of covalently modified peptides by Chien-Wen Hung; Andreas Schlosser; Junhua Wei; Wolf D. Lehmann (pp. 1003-1016).

Collision-induced reporter fragmentations of the currently most important covalent peptide modifications as detected by tandem mass spectrometry are summarized. These fragmentations comprise the formation of reporter ions, which are preferentially immonium ions, immonium ion-derived fragments or side chain fragments. In addition, the reporter neutral loss reactions for covalently modified amino acid residues are summarized. For each individual covalent modification which can be recognized by a reporter fragmentation, the accurate mass shift and the gross formula shift of the modified amino acid residue are given. The same set of data is provided for the reporter fragmentations. Finally, an extensive accurate mass and gross formula list is presented as supplementary material, describing mostly regular and modified y₁ and dipeptide a and b ions, which are helpful for identification of the peptide ends of covalently modified peptides. Figure When modified peptides are fragmented by collision-induced dissociation in a tandem mass spectrometer, the modification is either lost as part of a charged fragment, so that a reporter ion for the modification is generated or it is lost as part of a neutral fragment, so that a modification-specific reporter neutral loss is observed in the fragment ion spectrum.

Keywords: Peptide modifications; Covalent modifications; Tandem mass spectrometry; Neutral loss; Marker ions

Quantitative mass spectrometry in proteomics: a critical review by Marcus Bantscheff; Markus Schirle; Gavain Sweetman; Jens Rick; Bernhard Kuster (pp. 1017-1031).

The quantification of differences between two or more physiological states of a biological system is among the most important but also most challenging technical tasks in proteomics. In addition to the classical methods of differential protein gel or blot staining by dyes and fluorophores, mass-spectrometry-based quantification methods have gained increasing popularity over the past five years. Most of these methods employ differential stable isotope labeling to create a specific mass tag that can be recognized by a mass spectrometer and at the same time provide the basis for quantification. These mass tags can be introduced into proteins or peptides (i) metabolically, (ii) by chemical means, (iii) enzymatically, or (iv) provided by spiked synthetic peptide standards. In contrast, label-free quantification approaches aim to correlate the mass spectrometric signal of intact proteolytic peptides or the number of peptide sequencing events with the relative or absolute protein quantity directly. In this review, we critically examine the more commonly used quantitative mass spectrometry methods for their individual merits and discuss challenges in arriving at meaningful interpretations of quantitative proteomic data. Figure Common quantitative mass spectrometry workflows. Boxes in blue and yellow represent two experimental conditions. Horizontal lines indicate when samples are combined. Dashed lines indicate points at which experimental variation and thus quantification errors can occur. (adapted with permission from [11])

Keywords: Quantitative proteomics; Mass spectrometry; Stable isotope labeling

Two-dimensional electrophoresis of membrane proteins by Ralf J. Braun; Norbert Kinkl; Monika Beer; Marius Ueffing (pp. 1033-1045).

One third of all genes of various organisms encode membrane proteins, emphasizing their crucial cellular role. However, due to their high hydrophobicity, membrane proteins demonstrate low solubility and a high tendency for aggregation. Indeed, conventional two-dimensional gel electrophoresis (2-DE), a powerful electrophoretic method for the separation of complex protein samples that applies isoelectric focusing (IEF) in the first dimension and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) in the second dimension, has a strong bias against membrane proteins. This review describes two-dimensional electrophoretic techniques that can be used to separate membrane proteins. Alternative methods for performing conventional 2-DE are highlighted; these involve replacing the IEF with electrophoresis using cationic detergents, namely 16-benzyldimethyl-n-hexadecylammonium chloride (16-BAC) and cetyl trimethyl ammonium bromide (CTAB), or the anionic detergent SDS. Finally, the separation of native membrane protein complexes through the application of blue and clear native gel electrophoresis (BN/CN-PAGE) is reviewed, as well as the free-flow electrophoresis (FFE) of membranes.

Keywords: Membrane proteins; SDS/SDS-PAGE; 16-BAC/SDS-PAGE; BN-PAGE; Free-flow electrophoresis

Investigation of sample-purification procedures for MALDI-based proteomic studies by Marianne André; Michael Karas (pp. 1047-1053).

Purification methods for proteomics samples are of crucial concern for improving the quality of the sample delivered to the mass spectrometer. They constitute the link between the mass spectrometer and protein processing and peptide isolation steps that usually require solvents, buffers, or detergents completely incompatible with MS-analysis conditions. This work describes three new clean-up procedures using synthetic membranes and polymer media and compares them with standard procedures. The efficiency of each of the purification procedures was studied via application to four standards and two membrane proteins. This work highlights the importance of versatility in sample preparation, especially for MS-based proteomic investigations. Figure PMF spectra obtained after MALDI-TOF measurements of bovine mitochondrial complex III (A) and complex IV (B) in-solution digests, with and without purification

Keywords: MALDI mass spectrometry; Proteomics; Peptide purification

Evaluation of two proteomics technologies used to screen the membrane proteomes of wild-type Corynebacterium glutamicum and an L-lysine-producing strain by Daniela Schluesener; Matthias Rögner; Ansgar Poetsch (pp. 1055-1064).

The membrane proteomes of a wild-type Corynebacterium glutamicum and an L-lysine-producing strain were quantitatively analyzed by two complementary proteomics techniques—anion exchange chromatography AIEC/SDS-PAGE and 16BAC-PAGE/SDS-PAGE—and the results were compared. Although both techniques allow for the fast screening of differences in protein abundance, AIEC/SDS-PAGE was superior to 16BAC-PAGE/SDS-PAGE with respect to protein separation, it was more suitable for relative protein quantification, and allowed more differentially regulated proteins to be detected (the succinate dehydrogenase complex, an ABC-type cobalamin/Fe³⁺ siderophore transport system, the maltose binding protein, and a subunit of the cytochrome bc-aa₃ supercomplex were upregulated, while a periplasmic component of an ABC-type transporter and an iron-regulated ABC-type transporter were downregulated in the producer). The results indicate the important role of tricarboxylic acid cycle enzymes as well as the adaptation of transport processes in L-lysine-producing cells. Since the only genetic differences between the wild type and the L-lysine producer occur between four central metabolic enzymes in the cytoplasm, our study illustrates the complex effects of metabolic engineering on cell physiology and the power of the new AIEC/SDS-PAGE proteomics approach to detect these effects.

Keywords: Membrane proteins; Anion exchange chromatography; Mass spectrometry; Quantitative analysis

Miniaturized monolithic disks for immunoadsorption of cardiac biomarkers from serum by N. Delmotte; U. Kobold; T. Meier; A. Gallusser; A. Strancar; C. G. Huber (pp. 1065-1074).

Immunoadsorbers based on 2.0 × 6.0 mm i.d., epoxy-bearing, methacrylate-based monolithic disks were developed in order to target myoglobin and N-terminal pro-natriuretic peptide (NT-proBNP), two biomarkers involved in cardiovascular disease. In both cases, antibodies were successfully coupled to the polymeric disk material. The developed immunoadsorbers permitted the selective isolation of myoglobin and NT-proBNP from human serum. Myoglobin was successfully isolated and detected from serum samples at concentrations down to 250 fmol μL⁻¹. However, the affinity of the antibodies was not sufficient for the analysis of low-concentration clinical samples. Frontal analysis of anti-NT-proBNP disks revealed the ability of the immunoadsorber to bind up to 250 pmol NT-proBNP, which is more than sufficient for the analysis of clinical samples. Anti-NT-proBNP disks showed good stability over more than 18 months and excellent batch-to-batch reproducibility. Moreover, anti-NT-proBNP disks permitted the isolation of NT-proBNP at concentrations down to 750 amol μL⁻¹ in serum, corresponding to concentrations of strongly diseased patients. Using reversed-phase trapping columns, the detection of NT-proBNP eluted from immunoadsorbers by mass spectrometry was achieved for concentrations down to 7.8 fmol μL⁻¹.

Keywords: Immunoadsorber; Monolithic disk; Affinity chromatography; Liquid chromatography; Mass spectrometry; Myoglobin; N-terminal natriuretic peptide

Lung alveolar proteomics of bronchoalveolar lavage from a pulmonary alveolar proteinosis patient using high-resolution FTICR mass spectrometry by Yu Bai; Dmitry Galetskiy; Eugen Damoc; Jan Ripper; Markus Woischnik; Matthias Griese; Zhiqiang Liu; Shuying Liu; Michael Przybylski (pp. 1075-1085).

High-resolution Fourier transform ion cyclotron resonance (FTICR) mass spectrometry was developed and applied to the proteome analysis of bronchoalveolar lavage fluid (BALF) from a patient with pulmonary alveolar proteinosis. With use of 1-D and 2-D gel electrophoresis, surfactant protein A (SP-A) and other surfactant-related lung alveolar proteins were efficiently separated and identified by matrix-assisted laser desorption/ionization FTICR mass spectrometry . Low molecular mass BALF proteins were separated using a gradient 2-D gel. An efficient extraction/precipitation system was developed and used for the enrichment of surfactant proteins. The result of the BALF proteome analysis show the presence of several isoforms of SP-A, in which an N-non-glycosylierte form and several proline hydroxylations were identified. Furthermore, a number of protein spots were found to contain a mixture of proteins unresolved by 2-D gel electrophoresis, illustrating the feasibility of high-resolution mass spectrometry to provide identifications of proteins that remain unseparated in 2-D gels even upon extended pH gradients.

Keywords: Pulmonary alveolar proteinosis; Lung surfactant proteins; Bronchoalveolar lavage fluid; Fourier transform ion cyclotron resonance mass spectrometry; Proteome analysis

Proteome analysis of Apis mellifera royal jelly by Simone Schönleben; Albert Sickmann; Martin J. Mueller; Joerg Reinders (pp. 1087-1093).

Royal jelly plays a pivotal role in the development of honey bee larvae. However, while various health promoting properties of royal jelly have been reported, most of the active substances within royal jelly that lead to these properties are still unknown. Since up to 50% (dry mass) of royal jelly is protein, royal jelly proteome analysis is a promising starting point for attempts to identify the proteins that provide health-promoting effects. However, the comprehensive analysis of royal jelly proteins is hampered by the enormous abundance of some proteins in the major royal jelly protein family, which constitutes 80–90% of the royal jelly proteome. The high heterogeneity of these proteins is an additional challenge for proteomic analysis, since it necessitates the use of analytical techniques that provide high resolution and a wide dynamic range. The application of individual methods such as 2D-PAGE or multidimensional chromatography can only yield certain subpopulations of a proteome due to the specific bias of each method. We applied different methods for the prefractionation and separation of royal jelly proteins in order to circumvent the shortcomings of the individual techniques and achieve a high coverage of the royal jelly proteome. In this way, we were able to identify 20 different proteins in total, as well as to show a very high degree of cleavage of different proteins of the major royal jelly protein family. Furthermore, we investigated the protein phosphorylation of royal jelly proteins, and identified and located two phosphorylation sites within venom protein 2.

Keywords: Prefractionation; 2D-PAGE; Isoelectric focussing; Phosphorylation; Multidimensional liquid chromatography

Plasticity and robustness of protein patterns during reversible development in the honey bee (Apis mellifera) by Florian Wolschin; Gro V. Amdam (pp. 1095-1100).

With age, worker honey bees normally proceed from performing activities inside the nest to foraging in the field, creating an age-related division of labor. We previously established that the whole-body protein profiles of nest workers and foragers are different, and proposed that this proteomic divergence in part is explained by a shift in metabolic requirements as worker bees initiate intense flight. The unique plasticity of honey bee worker ontogeny, however, provides further opportunities to investigate if such changes in the proteome are dynamic or, alternatively, are permanently induced. Through manipulation of the social structure of colonies, foragers can be forced to revert to nest tasks, and in the current study we investigate how protein profiles respond to such reverse development. By using a quantitative LC-MS/MS-based approach in conjunction with robust statistical validation we show that after reversal from foraging to nest activities, subsets of proteins are detected at relative concentrations that characterize nest bees, whereas other proteins remain unchanged at relative concentrations normally found in foragers. In all, we quantified the levels of 81 proteins, and for 22 of these we found significant differences between worker groups before and after reversion. We interpret these patterns as examples of plasticity and robustness at the proteome level that are linked to characteristics of behavior and aging in Apis mellifera. Figure Quantitative LC-MS/MS in conjunction with robust statistical validation reveals plasticity and robustness of protein patterns during reversible development in the honey bee (Apis mellifera)

Keywords: Apis mellifera ; Proteomics; Protein quantification; Behavioral reversion; Aging

Molecular imaging by Reiner Salzer (pp. 1101-1102).

was Head of the Institute of Analytical Chemistry at the Department of Chemistry at Dresden University of Technology until his retirement in 2007. His research is focused on spectral imaging for fast monitoring, on polymers with biologically active functions and on electronic media in university education. He is a member of the label committee for the Chemistry Eurobachelor® and is also on the Advisory Board of Analytical and Bioanalytical Chemistry.

Multidimensional electrochemical imaging in materials science by Sabine Szunerits; Sascha E. Pust; Gunther Wittstock (pp. 1103-1120).

In the past 20 years the characterization of electroactive surfaces and electrode reactions by scanning probe techniques has advanced significantly, benefiting from instrumental and methodological developments in the field. Electrochemical and electrical analysis instruments are attractive tools for identifying regions of different electrochemical properties and chemical reactivity and contribute to the advancement of molecular electronics. Besides their function as a surface analytical device, they have proved to be unique tools for local synthesis of polymers, metal depots, clusters, etc. This review will focus primarily on progress made by use of scanning electrochemical microscopy (SECM), conductive AFM (C-AFM), electrochemical scanning tunneling microscopy (EC-STM), and surface potential measurements, for example Kelvin probe force microscopy (KFM), for multidimensional imaging of potential-dependent processes on metals and electrified surfaces modified with polymers and self assembled monolayers. Figure Electrochemical and electrical tools like scanning electrochemical microscopy, conductive atomic force microscopy, electrochemical scannig tunneling microscopy and Kelvin probe force microscopy (see figure) are powerful tools for the multidimensional imaging of potential-dependent processes on metals and electrified surfaces modified with polymers and self assembled monolayers.

Keywords: SECM; C-AFM; Polymer; Metal; Self assembled layers; Imaging

Imaging with spectroscopic micro-analysis using synchrotron radiation by D. Eichert; L. Gregoratti; B. Kaulich; A. Marcello; P. Melpignano; L. Quaroni; M. Kiskinova (pp. 1121-1132).

Recent developments of element-specific microscopy techniques using synchrotron radiation are opening new opportunities for the analytical investigation of various heterogeneous materials. This article provides a general description of the operational principles of different microscopes allowing chemical and structural imaging combined with micro-spot spectroscopic analysis. Several selected examples are used to illustrate the potential of the synchrotron-based methods in terms of imaging and chemical sensitivity for identification of spatial variations in the composition of morphologically complex and nano-structured inorganic and organic materials, including biological samples.

Keywords: X-ray spectroscopy; Interface / Surface analysis; Cell systems / Single cell analysis; IR spectroscopy; Chemical imaging; X-ray microscopy

Methodology for fiber-optic Raman mapping and FTIR imaging of metastases in mouse brains by Christoph Krafft; Matthias Kirsch; Claudia Beleites; Gabriele Schackert; Reiner Salzer (pp. 1133-1142).

The objectives of this study were to optimize the preparation of pristine brain tissue to obtain reference information, to optimize the conditions for introducing a fiber-optic probe to acquire Raman maps, and to transfer previous results obtained from human brain tumors to an animal model. Brain metastases of malignant melanomas were induced by injecting tumor cells into the carotid artery of mice. The procedure mimicked hematogenous tumor spread in one brain hemisphere while the other hemisphere remained tumor free. Three series of sections were prepared consecutively from whole mouse brains: dried, thin sections for FTIR imaging, hematoxylin and eosin-stained thin sections for histopathological assessment, and pristine, 2-mm thick sections for Raman mapping. FTIR images were recorded using a spectrometer with a multi-channel detector. Raman maps were collected serially using a spectrometer coupled to a fiber-optic probe. The FTIR images and the Raman maps were segmented by cluster analysis. The color-coded cluster memberships coincided well with the morphology of mouse brains in stained tissue sections. More details in less time were resolved in FTIR images with a nominal resolution of 25 μm than in Raman maps collected with a laser focus 60 μm in diameter. The spectral contributions of melanin in tumor cells were resonance enhanced in Raman spectra on excitation at 785 nm which enabled their sensitive detection in Raman maps. Possible reasons why metastatic cells of malignant melanomas were not identified in FTIR images are discussed. Figure FTIR image of 310 × 398 pixels obtained from an unstained, dried 10 μm tissue section of normal mouse brain. Colors represent the class assignments by cluster analysis

Keywords: Brain tumors; Tissue classification; Animal model; FTIR imaging; Raman mapping

Multivariate image analysis of a set of FTIR microspectroscopy images of aged bovine muscle tissue combining image and design information by A. Kohler; D. Bertrand; H. Martens; K. Hannesson; C. Kirschner; R. Ofstad (pp. 1143-1153).

In this paper we present an algorithm for analysing sets of FTIR microscopic images of tissue sections. The proposed approach allows one to investigate sets of many FTIR tissue images both with respect to sample information (variation from image to image) and spatial variations of tissues (variation within the image). The algorithm is applied to FTIR microscopy images of beef loin muscles containing myofibre and connective tissue regions. The FTIR microscopy images are taken of sub-samples from five different beef loin muscles that were aged for four different lengths of time. The images were investigated regarding variation due to the ageing length and due to the homogeneity of the connective tissue regions. The presented algorithm consists of the following main elements: (1) pre-processing of the spectra to overcome large quality differences in FTIR spectra and differences due to scatter effects, (2) identification of connective tissue regions in every image, (3) labelling of every connective tissue spectrum with respect to its location in the connective tissue region, and (4) analysis of variations in the FTIR microscopic images in regard to ageing time and pixel position of the spectra in the connective tissue region. Important spectral parameters characterising collagen and proteoglycan structure were determined. Figure Effective optical path length estimated by EMSC

Keywords: FTIR imaging; Pre-processing; Standardisation; Extended multiplicative signal correction; ANOVA PLSR; Hyperspectral imaging; PCA; Connective tissue

Towards a practical Fourier transform infrared chemical imaging protocol for cancer histopathology by Rohit Bhargava (pp. 1155-1169).

Fourier transform infrared (FTIR) chemical imaging is a strongly emerging technology that is being increasingly applied to examine tissues in a high-throughput manner. The resulting data quality and quantity have permitted several groups to provide evidence for applicability to cancer pathology. It is critical to understand, however, that an integrated approach with optimal data acquisition, classification, and validation is necessary to realize practical protocols that can be translated to the clinic. Here, we first review the development of technology relevant to clinical translation of FTIR imaging for cancer pathology. The role of each component in this approach is discussed separately by quantitative analysis of the effects of changing parameters on the classification results. We focus on the histology of prostate tissue to illustrate factors in developing a practical protocol for automated histopathology. Next, we demonstrate how these protocols can be used to analyze the effect of experimental parameters on prediction accuracy by analyzing the effects of varying spatial resolution, spectral resolution, and signal to noise ratio. Classification accuracy is shown to depend on the signal to noise ratio of recorded data, while depending only weakly on spectral resolution. Figure Correlation between conventionally stained and FTIR chemical images for pathology applications

Keywords: Fourier transform infrared spectroscopy; FTIR imaging; Infrared microscopy; Prostate; Histopathology; Microspectroscopy

Three-dimensional micro-XRF under cryogenic conditions: a pilot experiment for spatially resolved trace analysis in biological specimens by Birgit Kanngießer; Wolfgang Malzer; Marcel Pagels; Lars Lühl; Gundolf Weseloh (pp. 1171-1176).

Three-dimensional micro-XRF is a recently developed microprobe which facilitates three-dimensional resolved chemical analyses with a resolution of around 20 μm. Arbitrary sites or sections of samples can be investigated without the need to section specimens physically. In this paper we demonstrate the use of the microprobe in combination with a cold nitrogen gas stream for the cryogenic fixation of specimens. A 3D micro-XRF setup at the new microfocus beamline at BESSY II was equipped with a nitrogen cryogenic stream. The distribution of Ca, Fe, Zn and Cu across virtual cross sections of a water-rich sample, the root of common duckweed, could be investigated without further sample preparation. This paper demonstrates the capabilities of 3D micro-XRF under cryogenic conditions for investigations of biological specimens.

Keywords: 3D micro-XRF; Duckweed; Lemna minor ; Cryogenic cooling; Trace elements

In situ synthesis of molecularly imprinted polymers on glass microspheres in a column by Yan Zhuang; Hongpeng Luo; Deliang Duan; Lirong Chen; Xiaojie Xu (pp. 1177-1183).

A facile method to fabricate molecularly imprinted polymers (MIPs) on glass microspheres in a column was developed. The column was prepacked with glass microspheres, and then the prepolymerization mixture was injected into the interstitial volume of the column. The polymerization took place in situ and the column could be directly used for high-performance liquid chromatography after the template had been removed. The template consumption was reduced greatly because the prepolymerization mixture just filled the interstitial volume between the glass microspheres in the column. The MIPs obtained exhibited better kinetic properties, higher efficiency, and low back pressure of the column. Emodin imprinted polymers were prepared by this method and were used for solid-phase extraction.

Keywords: Molecularly imprinted polymer; In-column; Glass microsphere; Emodin; Solid-phase extraction

Characterization of nanoindentation-induced residual stresses in human enamel by Raman microspectroscopy by Li-Hong He; Elizabeth A. Carter; Michael V. Swain (pp. 1185-1192).

The objective of this research was to investigate nanoindentation-induced residual stresses in human enamel using Raman microspectroscopy and establish if this approach can be used as a stress meter. Healthy human premolars and sintered hydroxyapatite samples were embedded, cut, and the surfaces were polished finely with a 0.05 μm polishing paste before Berkovich and spherical indentations were made with a force of 100 mN. Spectra were collected using a Renishaw Raman InVia reflex microscope equipped with an air-cooled charge-coupled device (CCD) camera. Sample excitation was achieved using either an argon ion laser emitting at 514.5-nm or a NIR diode laser emitting at 830-nm. The residual micro stresses within and surrounding the indentation impressions were monitored by mapping the position of the ν₁(PO₄) band of (crystalline) hydroxyapatite. The Raman maps coincided well with the optical micrographs of the samples. Despite the presence of a fluorescence background from the organic component of human enamel, spectra collected using 514.5-nm excitation exhibited more significant shifts in the position of the ν₁(PO₄) band than spectra collected using 830-nm excitation. This implies that the former excitation may be a more appropriate excitation for stress detection. It was concluded that Raman microspectroscopy provides a novel high-resolution and non-destructive method for exploring the role of microstructure on the residual stress distribution within natural biocomposites. Figure Stress maps of nanoindentation impressions on both human enamel and hydroxyapatite disk via Raman Microspectroscopy

Keywords: Raman spectroscopy; Stress; Enamel; Hydroxyapatite

Detection of avian influenza virus using an interferometric biosensor by Jie Xu; David Suarez; David S. Gottfried (pp. 1193-1199).

An interferometric biosensor immunoassay for direct and label-less detection of avian influenza through whole virus capture on a planar optical waveguide is described. The assay response is based on index of refraction changes that occur upon binding of virus particles to unique antigen-specific (hemagglutinin) antibodies on the waveguide surface. Three virus subtypes (two H7 and one H8) in buffer solution were tested using both monoclonal and polyclonal capture antibodies. The real-time response of the antigen–antibody interaction was measured and was shown to be concentration-dependent, with detection limits as low as 0.0005 hemagglutination units per milliliter. A simple sandwich assay was shown to further increase the biosensor response. Figure Response of the optical interferometric biosensor (inset) to avian influenza virus using a monoclonal antibody (mAb) for whole virus capture in a direct binding assay. The response of the anti-H7 mAb is specific to the H7 subtype

Keywords: Optical waveguide; Interferometer; Immunoassay; Antibody; Hemagglutinin

Certification of SRM 1589a PCBs, pesticides, PBDEs, and dioxins/furans in human serum by Michele M. Schantz; Jennifer M. Keller; Stefan Leigh; Donald G. Patterson Jr; Katherine E. Sharpless; Andreas Sjödin; Heather M. Stapleton; Robert Swarthout; Wayman E. Turner; Stephen A. Wise (pp. 1201-1208).

The Certificate of Analysis for SRM 1589a PCBs, Pesticides, PBDEs, and Dioxins/Furans in Human Serum has been updated to include certified concentration values for 27 polychlorinated biphenyl (PCB) congeners, three chlorinated pesticides, and four polybrominated diphenyl ether (PBDE) congeners as well as reference concentration values for 27 additional PCB congeners, six additional chlorinated pesticides, three additional PBDE congeners, and selected polychlorinated dibenzo-p-dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs). This represents an addition of concentration values for 29 PCB congeners and for PBDE congeners that were not quantified in the previous issue of SRM 1589a. With the increased number of certified and reference concentration values for PCBs and the inclusion of certified and reference concentration values for PBDEs, this serum material will be more useful as a reference material for contaminant monitoring in human tissues and fluids.

Keywords: PCBs; Pesticides; PBDEs; Human serum; Human exposure

Development and validation of a qualitative screening method for the detection of exogenous anabolic steroids in urine by liquid chromatography-tandem mass spectrometry by Oscar J. Pozo; Peter Van Eenoo; Koen Deventer; Frans T. Delbeke (pp. 1209-1224).

A screening method for the urinary detection of 34 exogenous anabolic steroids has been developed. The method involves an enzymatic hydrolysis, liquid-liquid extraction and detection by liquid chromatography-tandem mass spectrometry. The use of some adducts such as [M+NH₄]⁺, [M+CH₃COO]⁻ and [M+H+MeOH]⁺ was necessary in order to detect some analytes at the required level (lower than 10 ng/ml). Two transitions were selected for each analyte. Different concentration factors have been studied in order to increase the sensitivity. A concentration factor of 50 was selected for the screening method although the high ion suppression observed under these conditions can hamper its application as a quantitative method. The method was validated and limits of detection were obtained by spiking ten different blank urine samples at five different concentration levels. Up to 29 analytes were detected in all spiked urines at the required level. Limits of detection between 1 and 10 ng/ml were obtained for most analytes which fulfil current requirements. The applicability of the method was shown by analysing positive samples.

Keywords: Anabolic steroids; Screening; Doping analysis; Metabolites; Mass spectrometry

New concept for a toxicity assay based on multiple indexes from the wave shape of damped metabolic oscillation induced in living yeast cells (part I): characterization of the phenomenon by H. Nakamura; M. Suzuki (pp. 1225-1232).

The damped glycolytic oscillation phenomenon occurring in starved cells of the yeast Saccharomyces cerevisiae (NBRC 0565) was characterization for application to a toxicity bioassay. S. cerevisiae was grown under semi-anaerobic conditions. The transient oscillations were observed photometrically as the time course of the fluorescent intensity of reduced pyridine nucleotide resulting from instantaneous addition of glucose to a cell suspension. In this study, simple and reproducible conditions inducing damped oscillations were obtained by modifying a literature method. For estimation of the wave shapes of the damped oscillations we used six indexes. To investigate the total reproducibility as the averaged relative standard deviation (RSD_av) for the six indexes obtained from the wave shapes, the damped oscillations were induced under the optimum conditions and the RSD_av values were calculated as 14% in a buffer cell suspension (n = 62) and 22% in a water cell suspension (n = 78). Finally, the effects of glucose concentration on the six indexes were examined, and all the indexes changed when the glucose concentration was changed. Excellent correlations were obtained between the index of oscillation-state time and the concentration of glucose in a buffer cell suspension (r = 0.9985, 0.5–250 mmol L⁻¹, 10 points) and in a water cell suspension (r = 0.9989, 2.5 μmol L⁻¹–250 mmol L⁻¹, 12 points), respectively. Figure Characterization of damped glycolytic oscillation, (a) typical shape, and (b) its estimation

Keywords: Characterized damped metabolic oscillation; Glycolysis; Yeast saccharomyces cerevisiae ; Intact cell toxicity bioassay; Multiple indexes

New concept for a toxicity assay based on multiple indexes from the wave shape of damped metabolic oscillation induced in living yeast cells (part II): application to analytical toxicology by H. Nakamura; M. Suzuki (pp. 1233-1241).

An ideal toxicity assay should utilize multiple indexes obtained from transient changes of metabolic activities. Here, we demonstrate the possibility for a novel toxicity bioassay using the damped glycolytic oscillation phenomenon occurring in starved yeast cells. In a previous study, the phenomenon was characterized in detail. Under optimum conditions to induce the phenomenon, the wave shapes of the damped glycolytic oscillations were changed by the instantaneous addition of both glucose and chemicals and by changing the chemical concentration. We estimated the changes in the oscillation wave shapes as six indexes, i.e., the number of wave cycles, maximum amplitude, oscillation frequency, attenuation coefficient, initial peak height, and non-steady-state time. These index changes were obtained from several kinds of chemicals. The chemicals, especially those for acids (0.01–100 mM HCl and 0.01–50 mM citric acid), bases (0.001–50 mM KOH), heavy metal ions (1–1,000 mg L⁻¹; Cu²⁺, Pb²⁺, Cd²⁺, Hg²⁺), respiratory inhibitors (3–500 mg L⁻¹ NaN₃), dissolved oxygen removers (10–300 mg L⁻¹ NaSO₃), surfactants (10–200 mg L⁻¹ benzalkonium chloride), and aldehyde (10–1,000 mg L⁻¹ acetaldehyde), showed characteristic patterns depending on each chemical and its concentration. These significant results demonstrate the possibilities of new methods for both toxicity qualification and quantification. Figure Influences of surfactant on the oscillation wave shape

Keywords: Damped glycolytic oscillation; Yeast Saccharomyce cerevisiae ; Living cell toxicity bioassay; Multiple indexes; Pattern analysis

In vitro and in vivo metabolisms of K-48 by B. Benkő; H. Kalász; K. Ludányi; G. Petroianu; K. Kuca; F. Darvas; K. Tekes (pp. 1243-1247).

Metabolic pathways of the oxime K-48 have been elucidated by means of in vitro and in vivo experiments. K-48 exposure to rat liver microsomal fraction resulted in the formation of a hydroxylated derivative, in addition to a small molecular fragment. The in vivo metabolism in rats was investigated after intramuscular administration of 50 μmol oxime. K-48 was present in the rat serum in unchanged form. Similarly, the analysis of rat cerebrospinal fluid indicated the sole occurrence of unchanged K-48. In contrast, unchanged K-48 was not found in the rat urine, where only the metabolite generated by epoxidation on the alkyl chain connecting the two pyridinium rings was present. The presence of unchanged K-48 in the serum and cerebrospinal fluid facilitates quantitative determination using HPLC separation and ultraviolet absorbance detection. Figure Suggested metabolic pathways of K-48

Keywords: Microsomal metabolism; Cholinesterase reactivator; K-48; In vivo metabolism; HPLC–MS

Structural and analytical studies of silica accumulations in Equisetum hyemale by Lanny Sapei; Notburga Gierlinger; Jürgen Hartmann; Robert Nöske; Peter Strauch; Oskar Paris (pp. 1249-1257).

Horsetail (Equisetum spp.) is known as one of the strongest accumulators of silicon among higher terrestrial plants. We use the combination of position-resolved analytical techniques, namely microtomography, energy-dispersive X-Ray elemental mapping, Raman microscopy, as well as small-angle and wide-angle scattering of X-rays, to study the type, distribution and nanostructure of silica in the internodes of Equisetum hyemale. The predominant silicification pattern is a thin continuous layer on the entire outer epidermis with the highest density in particular knob regions of the long epidermal cells. The knob tips contain up to 33 wt% silicon in the form of pure hydrated amorphous silica, while the silica content is lower in the inner part of the knobs and on the continuous layer. In contrast to the knob tips, the silica in these regions lacks silanol groups and is proposed to be in close association with polysaccharides. No mentionable amount of crystalline silica is detected by wide-angle X-ray scattering. The small-angle X-ray scattering data are consistent with the presence of colloidal, sheet-like silica agglomerates with a thickness of about 2 nm. From these results we conclude that there are at least two distinct forms of silica in E. hyemale which may have different functions. The close association of silica with cell wall polymers suggests that they may act as a polymeric template that controls the shape and size of the colloidal silica particles similar to many other biominerals and mineralised tissues. We propose that owing to its specific distribution in E. hyemale, a protective role and possibly also an important biomechanical role are among the most likely functions of silica in these plants. Figure 3D rendering of X-ray microtomography data from a dry Equisetum hyemale stalk. The red colour indicates high X-ray absorption values due to local silica accumulations

Keywords: Equisetum hyemale ; Silica; Raman microscopy; Microtomography; Small-angle X-ray scattering; Wide-angle X-ray scattering

Investigation of cuticular hydrocarbons from Bagrada hilaris genders by SPME/GC-MS by C. De Pasquale; S. Guarino; E. Peri; G. Alonzo; S. Colazza (pp. 1259-1265).

The cuticular hydrocarbons of male and female Bagrada hilaris Burmeister (Heteroptera: Pentatomidae) were investigated, by headspace solid-phase microextraction followed by gas chromatography–mass spectrometry. Measurements were done with fiber coatings of different polarity after optimization of headspace volumes and extraction temperatures. This resulted in the use of polyacrylate fiber, 22-ml vial as the sample holder, and an extraction temperature of 150 °C. The analytical procedures allowed identification of 13 peaks, corresponding to a homologous series of n-alkanes (nC₁₇–nC₂₉). The hydrocarbon profiles of male and female B. hilaris were qualitatively equal, but marked sex-specific quantitative differences were observed for some of the linear alkanes.

Keywords: Bagrada hilaris ; Painted bug; Headspace solid-phase microextraction; Cuticular hydrocarbons; Gas chromatography–mass spectrometry

Benchmarking and validating algorithms that estimate pK _a values of drugs based on their molecular structures by Milan Meloun; Sylva Bordovská (pp. 1267-1281).

The REGDIA regression diagnostics algorithm in S-Plus is introduced in order to examine the accuracy of pK _a predictions made with four updated programs: PALLAS, MARVIN, ACD/pKa and SPARC. This report reviews the current status of computational tools for predicting the pK _a values of organic drug-like compounds. Outlier predicted pK _a values correspond to molecules that are poorly characterized by the pK _a prediction program concerned. The statistical detection of outliers can fail because of masking and swamping effects. The Williams graph was selected to give the most reliable detection of outliers. Six statistical characteristics (F _exp, R ², $$ { ext{R}}^{2}_{{ ext{P}}} $$ , MEP, AIC, and s(e) in pK _a units) of the results obtained when four selected pK _a prediction algorithms were applied to three datasets were examined. The highest values of F _exp, R ², $$ { ext{R}}^{2}_{{ ext{P}}} $$ , the lowest values of MEP and s(e), and the most negative AIC were found using the ACD/pK _a algorithm for pK _a prediction, so this algorithm achieves the best predictive power and the most accurate results. The proposed accuracy test performed by the REGDIA program can also be applied to test the accuracy of other predicted values, such as log P, log D, aqueous solubility or certain physicochemical properties of drug molecules.

Keywords: pK _a prediction; pK _a accuracy; Dissociation constants; Outliers; Influential points; Residuals; Goodness-of-fit; Williams graph

Electrocatalytic oxidation of dopamine at an ionic liquid modified carbon paste electrode and its analytical application by Wei Sun; Maoxia Yang; Kui Jiao (pp. 1283-1291).

A room-temperature ionic liquid N-butylpyridinium hexafluorophosphate was used as a binder to construct an ionic liquid modified carbon paste electrode, which was characterized by scanning electron microscopy and electrochemical impedance spectroscopy. The ionic liquid carbon paste electrode (IL-CPE) showed enhanced electrochemical response and strong analytical activity towards the electrochemical oxidation of dopamine (DA). A pair of well-defined quasireversible redox peaks of DA appeared, with the redox peaks located at 215 mV (E _pa) and 151 mV (E _pc) (vs. the saturated calomel electrode, SCE) in pH 6.0 phosphate buffer solution. The formal potential (E ^0´) was calculated as 183 mV (vs. SCE) and the peak-to-peak separation as 64 mV. The electrochemical behavior of DA on the IL-CPE was carefully investigated. Under the optimal conditions, the anodic peak currents increased linearly with the concentration of DA in the range 1.0 × 10⁻⁶–8.0 × 10⁻⁴ mol/L and the detection limit was calculated as 7.0 × 10⁻⁷ mol/L (3σ). The interferences of foreign substances were investigated and the proposed method was successfully applied to the determination of DA injection samples. The IL-CPE fabricated was sensitive, selective and showed good ability to distinguish the coexisting ascorbic acid and uric acid.

Keywords: Dopamine; Room-temperature ionic liquid; N-Butylpyridinium hexafluorophosphate; Carbon paste electrode; Electrooxidation

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Analytical and Bioanalytical Chemistry (v.389, #4)

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Analytical and Bioanalytical Chemistry (v.389, #4)