New method for prefractionation of plasma for proteomic analysis

The depth of proteome analysis is severely limited in complex samples with a wide dynamic range of protein abundance such as plasma. Removal of high‐abundance proteins should reveal the signal of lower abundance plasma proteins. However, smaller proteins may be part of larger protein complexes and hence the removal of proteins involved in complexes with high‐abundance proteins such as albumin may inhibit the search for disease biomarkers. Prefractionation of a sample divides it into fractions of reduced complexity, allowing improved detection of lower abundance proteins. Using a prefractionation device, which employs Gradiflow? technology, we were able to separate small volume plasma samples into multiple fractions based on the molecular weight and/or charge. The resulting samples of reduced complexity were directly compatible with 2‐DE. The use of this prefractionation machine may therefore be useful in the hunt for disease biomarkers.     

Comparative analysis of proteomic changes in contrasting flax cultivars upon cadmium exposure

Cadmium (Cd) is classified as a serious pollutant due to its high toxicity, high carcinogenicity, and widespread presence in the environment. Phytoremediation represents an effective low‐cost approach for removing pollutants from contaminated soils, and a crop with significant phytoremediation potential is flax. However, significant differences in Cd accumulation and tolerance were previously found among commercial flax cultivars. Notably, cv. Jitka showed substantially higher tolerance to elevated Cd levels in soil and plant tissues than cv. Tábor. Here, significant changes in the expression of 14 proteins (related to disease/defense, metabolism, protein destination and storage, signal transduction, energy and cell structure) were detected by image and mass spectrometric analysis of two‐dimensionally separated proteins extracted from Cd‐treated cell suspension cultures derived from these contrasting cultivars. Further, two proteins, ferritin and glutamine synthetase (a key enzyme in glutathione biosynthesis), were only up‐regulated by Cd in cv. Jitka, indicating that Cd tolerance mechanisms in this cultivar may include maintenance of low Cd levels at sensitive sites by ferritin and low‐molecular weight thiol peptides binding Cd. The identified changes could facilitate marker‐assisted breeding for Cd tolerance and the development of transgenic flax lines with enhanced Cd tolerance and accumulation capacities for phytoremediating Cd‐contaminated soils.     

A statistical analysis of polysilicon etching in a chlorine-argon plasma

The effects of base pressure, etch pressure, gas flow rates of HCl, Cl₂, and argon, hexode temperature, DC self-bias, initial polysilicon thickness, and percent of overetch on the etch performance of polysilicon are examined. Statistical design of the experiments provided linear and quadratic models of the etch performance in terms of the aforementioned variables. These models were used to determine the relative importance of each process variable on the etch performance. Optical emission data were utilized as a means of endpoint detection and as a monitor of etch activity. The results indicate that the etch performance is more responsive to variations in physical mechanisms as opposed to chemical processes within the variable ranges used in these experiments.     

HPLC determination of berberine in plasma of patients with ischemic heart failure

Summary Berberine absorption by patients with ischemic heart failure (IHF) after oral administration and the relationship between clinical effect and plasma berberine concentration are studied. Plasma samples were pretreated by chloroform extraction. Berberine was determined on a μBondapak column with acetonitrile-phosphoric acid mobile phase and UV detection. The limit for berberine in plasma was 8 ng mL⁻¹ for an injection volume of 50 μL. Average berberine recovery was 96.5%. Results showed that improvements in symptoms were more significant for patients with plasma berberine concentration>0.1 mg L⁻¹ than for those with<0.1 mg L⁻¹. Plasma berberine monitoring may be helpful in the treatment of patients with IHF.     

Comparative proteomic analysis of B. cenocepacia using two-dimensional liquid separations coupled with mass spectrometry

Burkholderia cenocepacia is an important respiratory pathogen in persons with cystic fibrosis. We compared the proteomes of clinical and environmental isolates of B. cenocepacia by using a 2D liquid separation method coupled with mass spectrometry. Proteome maps of four B. cenocepacia isolates were generated. In the first dimension, 5 mg of protein from each isolate was fractionated by chromatofocusing (CF) in the range of pH 4.0-7.0. In the second dimension, each CF fraction was separated by NPS-RP-HPLC. Results of the 2D liquid separation were visualized as 2D UV maps, which allowed direct comparison of proteomes with high resolution and reproducibility. From the proteomic comparison of the four isolates, 38 of 96 differentially abundant proteins were identified by peptide mass fingerprinting and MS/MS sequence analysis using a partially annotated B. cenocepacia protein database. Many of the identified proteins in the clinical isolates are involved in gene translation and bacterial virulence such as transmissibility, resistance, and quorum sensing.     

Thermogravimetric analysis of coke formation on plasma treated Mo-Fe/HZSM-5 catalyst during non-oxidative aromatization of methane

Summary Thermogravimetric (TG) analysis confirms that glow discharge plasma treatment has a remarkable influence on coke formation during the non-oxidative aromatization of methane over a Mo-Fe/HZSM-5 catalyst. The plasma treatment favors formation of active carbon species for methane aromatization.</o:p>     

Automated analysis of terbutaline (BricanylR) in human plasma with liquid chromatography and electrochemical detection using column-switching (multidimensional chromatography)

Summary An automated method based on liquid chromatography has been developed for the determination of terbutaline in human plasma in the range of 5–50 pmole·ml^–1. The necessary sensitivity and selectivity was obtained by using electrochemical detection and a microprocessor-controlled column switching system. A combination of three columns was used: a C₈ type for pre-separation, a C₁₈ type for trapping and, for final separation, a strongly acidic ion exchanger. The accuracy of the method was examined by comparison with a method based on gas chromatography — mass spectrometry. The overall precision was ±3.5% and ±2.2% respectively at 5 and 50 pmole·ml^–1. The total absolute recovery for terbutaline and internal standard at the above concentration levels were in the range 85–106%.Presented at the 14th International Symposium on Chromatography London, September, 1982     

Development and application of a new on-line SPE system combined with LC-MS/MS detection for high throughput direct analysis of pharmaceutical compounds in plasma

A technique using a fully automated on-line solid phase extraction (SPE) system (Symbiosis, Spark Holland) combined with liquid chromatography (LC)-mass spectrometry (MS/MS) has been investigated for fast bioanalytical method development, method validation and sample analysis using both conventional C18 and monolithic columns. Online SPE LC-MS/MS methods were developed in the automated mode for the quantification of model compounds (propranolol and diclofenac) directly in rat plasma. Accuracy and precision using online SPE LC-MS/MS with conventional C18 and monolithic columns were in the range of 88-111% and 0.5-14%, respectively. Total analysis cycle time of 4 min per sample was demonstrated using the C18 column. Monolithic column allowed for 2 min total cycle time without compromising the quality and validation criteria of the method. Direct plasma sample injection without on-line SPE resulted in poor accuracy and precision in the range of 41-108% and 3-81%. Furthermore, the increase in back pressure resulted in column damage after the injection of only 60 samples.     

Interference-free determination of fluoroquinolone antibiotics in plasma by using excitation-emission matrix fluorescence coupled with second-order calibration algorithms

Fluoroquinolones or so-called second-generation quinolones, in particular, ofloxacin (OFL), norfloxacin (NOR), and enoxacin (ENO), with therapeutic advantages possess strongly overlapped fluorescence spectra. In this paper, two strategies were proposed for simultaneous direct determination of OFL, NOR and ENO in plasma by combining fluorescence excitation-emission matrix (EEM) with second-order calibration based on the alternating trilinear decomposition algorithm (ATLD) and parallel factor analysis (PARAFAC). The results showed that both algorithms could solve the problem of serious fluorescence spectral overlapping of the sought-for analytes even in the presence of uncalibrated interferents. However, ATLD has advantages of being insensitive to overestimated component number and fast convergence. The results by using ATLD with an estimated component number of five were reasonably acceptable for clinical analysis. The average recoveries of OFL, NOR and ENO in synthetic samples were 99.7 ± 2.4, 101.5 ± 2.4 and 97.3 ± 3.8%, respectively; the average recoveries of OFL, NOR and ENO in complex plasma were 94.3 ± 2.6, 85.6 ± 3.3 and 103.3 ± 3.0%, respectively.     

Simultaneous analysis of cocaine,benzoylecgonine, methylecgonine,and ecgonine in plasma using an exchange resin and gas chromatography-mass spectrometry

Summary A gas chromatographic-mass spectrometric method is described for the simultaneous analysis of cocaine and the hydrolytic products benzoylecgonine, methylecgonine and ecgonine from plasma (0.25–2 g/ml). Isopropylecgonine was incorporated as an internal standard. Samples were extracted using a sulfonate cation exchange resin, then derivatized with pentafluoropropionic anhydride and pentafluoropropanol. Analytical separations were on a dimethylsilicone capillary column using a temperature program, and detection was by selected ion monitoring of the electron impact generated fragments m/z 94, 182, 210, and 300.Presented at the poster session of American Association of Pharmaceutical Scientists 1991 Southeast Regional Meeting, Charleston, South Carolina, USA, April 4–5     

Comparative proteomic analysis of different Toxoplasma gondii genotypes by two‐dimensional fluorescence difference gel electrophoresis combined with mass spectrometry

Toxoplasma gondii is a protozoan parasite infecting almost all warm‐blooded animals and humans. There are three infective stages of T. gondii: the tachyzoites, the bradyzoites, and the oocysts. The tachyzoite is a rapidly multiplying stage and the main pathogenic factor. In North America and Europe, T. gondii is consisted of four major clonal lineages (namely Types I, II, III, and Type 12). In this study, we explored the proteomic profiles of different genotypes (Type I‐RH strain, Type II‐PRU strain, Type II‐TgQHO strain, and ToxoDB 9‐TgC7 strain) of T. gondii tachyzoites by using 2D DIGE combined with MALDI‐TOF MS. Totally, 110 differentially abundant protein spots were selected. Of these, 98 spots corresponding to 56 proteins from T. gondii were successfully identified. These included surface antigen (SAG1), heat shock protein 70 (Hsp 70), disulfide isomerase, coronin, heat shock protein 60 (Hsp 60), pyruvate kinase, receptor for activated C kinase 1, and peroxiredoxin. Gene ontology enrichment analysis revealed that most of the differentially abundant proteins were involved in biological regulation, metabolic process, response to stress, binding, antioxidant activity, and transporter activity. According to the KEGG metabolic pathway maps of T. gondii, some identified proteins were involved in the glycolytic/gluconeogenesis pathway. The present study identified differentially abundant proteins among different genotypes of T. gondii and these findings have implications for the better understanding of the phenotypic differences among the examined T. gondii genotypes, which in turn may contribute to the better control of toxoplasmosis.     

Comparative proteomic analysis using 2DE‐LC‐MS/MS reveals the mechanism of Fuzhuan brick tea extract against hepatic fat accumulation in rats with nonalcoholic fatty liver disease

Fuzhuan brick tea has received increasing attention in recent years owing to its benefits for nonalcoholic fatty liver disease (NAFLD) and associated metabolic syndrome. For exploring the ameliorative mechanism, the liver proteomes from three groups of rats fed either a normal control diet (NCD), a high fat diet (HFD), or a HFD supplemented with high‐dose Fuzhuan brick tea extract (FTE) (HFD + HFTE) were comprehensively compared by quantitative proteomics using 2DE‐LC‐MS/MS. This is the first study of the effects of tea aqueous extract on the liver proteome of rats suffering from metabolic syndrome. The results showed that 57 proteins displayed more than 1.5‐fold differences in at least one of two comparisons of HFD versus NCD and HFD versus HFD + HFTE due to HFD feeding and FTE treatment, respectively. Of them, over 75% of proteins exhibited a similar tendency of expression in the two comparisons, meaning FTE was able to correct HFD effects on rat livers. By function analyses, an extensive list of proteins was involved in sugar and lipid metabolism. Compared with HFD‐fed rats, the reduced lipogenesis and enhanced β‐oxidation, tricarboxylic acid cycle and respiratory chain in HFD + HFTE‐fed rats, which mainly contributed to ameliorate hepatic fat accumulation and associated NAFLD. Additionally, some putative drug targets were also revealed such as COX2, PGAM1, ACACB, FAS, and ECHS1.     

Evaluation of a rapid semi-quantitative analysis approach using inductively coupled plasma optical emission spectrometry with axial viewing

This work describes a rapid screening procedure for simultaneous determination of multiple elements by inductively coupled plasma optical emission spectrometry with axial viewing. The process requires no calibration reference solutions, and therefore requires a shorter analysis time per sample. The “Rapid Quant for Axial” worksheet contains stored calibration functions that are not adjusted between analyses. These functions were applied to the analysis of standard reference materials to demonstrate the effectiveness of the screening tool. Quantitative recoveries were obtained for most elements in a variety of biological materials. Aluminum, Cu, Mn, P, and Zn were determined in spinach leaves, for example, with errors ranging from − 45% (Al) to + 23% (Cu). A simulation of the worksheet procedure was also tested. With this technique, a single user-specific, multi-element reference solution was used to prepare a new set of calibration parameters to replace those stored in the worksheet. This procedure proved to be useful for sample screening when the analytes of interest were not present in the original calibration file, or when their concentrations differed significantly from those previously stored.     

In‐depth insight into the methods of plasma protein‐drug interaction studies: Comparison of capillary electrophoresis‐frontal analysis,isothermal titration calorimetry,circular dichroism and equilibrium dialysis

Plasma protein‐drug binding assays are routinely performed during the early stages of drug discovery and development, which creates demand for an automated high‐throughput screening assay to increase laboratory efficiency. A comprehensive comparison of the four methods typically used for determining the binding parameters is presented in this study with respect to the above demand. Capillary electrophoresis‐frontal analysis, isothermal titration calorimetry, circular dichroism and equilibrium dialysis were used to study the affinity of human serum albumin for diclofenac and lidocaine. These model drugs were chosen due to their different physico‐chemical properties and different binding sites on the albumin molecule, also resulting in different binding strength. The binding parameters estimated under the conditions as similar as possible were comparable among all these approaches as well as to the literature values. Besides this, the comparison of the results and especially other considerations demonstrated the benefits and drawbacks of the selected methods, with capillary electrophoresis‐frontal analysis being the best candidate for such studies.     

Chemical analysis by inductively coupled plasma atomic emission spectrometry of semiconducting ceramics of barium titanate doped with various metal oxides

The Ba and Ti macroconstituents as well as the impurities and dopants content (Al, Ca, Co, Cr, Cu, Fe, K, Li, Mg, Mn, Na, Nb, Ni, P, Pb, Si, Sr, W, Zn and Zr) in a dense (> 98% theoretical) barium titanate sample have been determined by inductively coupled plasma-atomic emission spectrometry after one of these decomposition routes: (a) decomposition with HCl in a PTFE-lined pressure vessel, (b) fusion with Na₂CO₃ in a platinum crucible, and (c) fusion with Li₂B₄O₇ in a graphite crucible. Matrix effects were taken into account. Detection limits for minors and trace elements were determined. High sensitivity and good precision were attained.Presented in part at the 1989 European Winter Conference on Plasma Spectrochemistry, Reutte, Austria     

Chemometric classification of gunshot residues based on energy dispersive X-ray microanalysis and inductively coupled plasma analysis with mass-spectrometric detection

A gunshot residue sample that was collected from an object or a suspected person is automatically searched for gunshot residue relevant particles. Particle data (such as size, morphology, position on the sample for manual relocation, etc.) as well as the corresponding X-ray spectra and images are stored. According to these data, particles are classified by the analysis-software into different groups: ‘gunshot residue characteristic’, ‘consistent with gunshot residue’ and environmental particles, respectively. Potential gunshot residue particles are manually checked and – if necessary – confirmed by the operating forensic scientist.