Mass spectrometric identification of proteins from silver-stained polyacrylamide gel: a method for the removal of silver ions to enhance sensitivity

Mass spectrometry is a powerful technique for the identification of proteins at nanogram quantities. However, some degree of sample preparation prior to mass spectrometry is required, and silver-stained protein gel samples are most problematic. Here we report our strategy to obtain peptide mass profiles from silver-stained protein gel samples from one- or two-dimensional gels by destaining prior to enzymatic digestion. This study demonstrates that by using the destaining method, the sensitivity and quality of mass spectra is increased for matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometric analysis, permitting more proteins to be identified by peptide mass database analysis.     

Automatic scanning of chromatograms by multi-scaling with a gamma-ray spectrometer

The location of gamma-emitting isotopes on Chromatographic thin layers is achieved, together with qualitative and quantitative analysis, by using only a gamma-ray spectrometer. This technique is applied to the determination of the Chromatographic behaviour of nanogram amounts of ⁵¹Cr, ⁵⁴Mn, ⁵⁹Fe, ⁶⁰Co, ⁶⁵Zn and ¹⁰⁹Cd thiocyanate complexes on silica gel. Separations are made of Co, Zn and Cd from Cr, Mn and Fe.     

Ultrasensitive and Simultaneous Determination of Arsenic and Antimony in Clinical Samples by Atomic Fluorescence Spectrometry

The determination of trace elements in human fluids facilitates the identification of metabolic disorders and physiological abnormities in clinical diagnosis. Elemental concentrations in serum, urine, and saliva are often low and highly prone to contamination. Consequently, a simple and ultrasensitive detection technique is required. In this study, a method was developed for the simultaneous determination of trace arsenic and antimony in clinical samples by hydride generation atomic fluorescence spectrometry. The experimental parameters that affect the generation, delivery, and atomization of volatile arsenic and antimony hydrides were investigated. The limits of quantification were calculated to be 0.0037 nanogram per milliliter and 0.0070 nanogram per milliliter for arsenic and antimony, respectively. The method was successfully applied for the analysis of fortified human serum, urine, saliva, and certified reference materials. The results obtained by the reported method were not statistically different from the certified values at the 95 percent confidence level.     

Photochemical analysis studies-III A fluorimetric and ultraviolet spectrophotometric study of the photolysis of chloroquine on silica-gel thin layers, and its analytical application

The photolysis of chloroquine on silica-gel thin layers has been studied by ultraviolet spectrophotometry and fluorimetry. It has been shown that a reversible photoisomerization of chloroquine takes place and yields a fluorescent photoproduct. The effect of pH, alkali-metal halides, and the eluent system on the fluorescence intensity of chloroquine and its photoproduct was investigated. The fluorescence on silica-gel thin layers is quenched by high concentrations of sodium hydroxide; this is explained in terms of interference of sodium ions in the hydrogen-bonded adsorption of chloroquine on the silica gel. The results have been used to develop a method for determination and separation of chloroquine at the nanogram level. Linear calibration plots are obtained, covering the range between 4 and 10(4) ng.     

Investigation of adsorption of nanogram quantities of iron(II) tris-(1,10-phenanthrolinate) on glasses and silica by thermal lens spectrometry

Thermal lens spectrometry is used for studying adsorption equilibria in aqueous solutions at the level of nanogram quantities of iron(II) tris-(1,10-phenanthrolinate) as a model system. The kinetics of the sorption of the chelate on silica is studied and adsorption isotherms are built. Thermal lensing is used as a method for direct determination of the chelate concentration adsorbed on a quartz surface. The detected amount is 4.1×10⁻¹⁵ mol at the area irradiated by the excitation beam. The adsorption of iron(II) tris-(1,10-phenanthrolinate) on laboratory glassware at the nanogram level is characterised by measuring the residual concentration of the sorbate in solution. A procedure for handling and cleaning the laboratory glassware for determining nanogram amounts of iron in aqueous solutions is proposed. The sensitivity of thermal lensing both in measuring adsorption on silica and glass and quartz surfaces is 100-fold higher than diffuse-reflectance measurements under the same conditions.     

A sensitive, rapid and specific technique for the detection of collagenase using zymography

A fast and specific method for the detection of collagenase by electrophoresis is described. The method avoids inclusion of the substrate in the resolving gel and can detect nanogram levels of the enzyme.     

Circular dichroism detection of optically active compounds in gas chromatography using vacuum ultraviolet synchrotron radiation

Circular dichroism has been employed as a detection technique in gas chromatography for specific monitoring of optically active compounds which absorb in the vacuum ultraviolet region. The synchrotron radiation from U9A beamline of National Synchrotron Light Source, Brookhaven National Laboratory, was used as the light source. The detection limit established using this system is at the nanogram level for a selected group of hydrocarbons.     

Identifizierung von Diphenylquecksilber,Phenylquecksilberchlorid und Quecksilber (II)-chlorid im Nanogrammbereich durch Dünnschicht-Chromatographie

Zusammenfassung Die genannten Substanzen werden auf Silicagel-Platten getrennt. Im Mikrogrammbereich werden sie durch Behandeln mit Joddampf nachgewiesen. Verwendet man die entsprechenden radioaktiv markierten Substanzen, lassen sie sich noch im Subnanogrammbereich durch Autoradiographie oder Flüssigszintillationszählung nachweisen.

---

Identification of diphenylmercury, phenylmercuric chloride, and mercuric chloride at the nanogram level by thin-layer chromatography

Summary The substances mentioned are separated on silica gel plates. In the microgram range they are detected by treating with iodine vapour. When radioactively labelled these substances may be detected even at the sub-nanogram level by autoradiography or liquid scintillation counting.

     

Identification of some polycyclic nitrogen-containing compounds in coal-derived oil

A systematic method for the identification of aza-arenes in coal-derived oil was developed. The basic nitrogen-containing substances were extracted with 6 M hydrochloric acid and fractionated sequentially by using gel chromatography and thin-layer chromatography (TLC) on an alumina plate. The aza-arenes in these fractions were separated by using glass capillary gas chromatography. Individual compounds in the column effluent were trapped in a system consisting of a valve for flow switching and a trapping tube made from a glass capillary. The fluorescence spectra of nanogram to subnanogram amounts of trapped compounds were measured. Some attempts were made to identify components based on their TLC RF values and their fluorescence spectra, in addition to their mass spectra.     

Electron Capture Gas Chromatography of Tertiary Amines After a Demethylation Reaction to Secondary Amines

Abstract

A gas chromatographic method for the determination of tertiary amines in micro- and nanogram amounts is presented. The tertiary amine is reacted with ethyl chloroformate to form a urethan, which is cleaved to a secondary amine. This is transformed to heptafluoro-butyramide, which makes quantitative determination with electron capture detection possible. The yields of the secondary amines for two tertiary amines were 60 and 75%. By use of internal standard technique in the entire reaction recoveries of 97.6 ± 2.2% at the 100 μg level and 100 ± 4% at the 200 ng level were obtained.     

Catalytic Hydrolysis of Urea Herbicides Combined with Column Chromatography

Abstract

A method is described for the hydrolysis of urea herbicides on silica gel surfaces, making use of the acidic silanol groups. Only a heating step is needed without reagents being added and complete hydrolysis to the corresponding anilines can be achived in about 15 min at 165°C. Gas and liquid chromatographic techniques coupled with derivatization of the anilines before and after the column are then introduced as the actual analysis step. The potential of these analytical systems is demonstrated with a residue analysis in soil. The selectivity and sensitivity (detection limits, low nanogram to low picogram level) permit quantitation of urea herbicide traces with a minimum of sample clean-up.     

Universal method for synthesis of artificial gel antibodies by the imprinting approach combined with a unique electrophoresis technique for detection of minute structural differences of proteins, viruses and cells (bacteria). Ib. Gel antibodies against proteins (hemoglobins)

Using the molecular imprinting approach, we have shown that polyacrylamide-based artificial antibodies against human and bovine hemoglobin have a very high selectivity, as revealed by the free-zone electrophoresis in a revolving capillary. By the same technique we have previously synthesized gel antibodies not only against proteins but also against viruses and bacteria. The synthesis is thus universal, i.e., it has the great advantage of not requiring a modification - or only a slight one - for each particular antigen. The combination synthesis of artificial gel antibodies and electrophoretic analysis reveals small discrepancies in shape and chemical composition not only of proteins, as shown here and in paper Ia, but also of viruses and bacteria, to be illustrated in papers II and III in this series. Upon rehydration, the freeze-dried gel antibodies, selective for human hemoglobin, regain their selectivity. The gel antibodies can repeatedly be used following the removal of the antigen (protein in this study) from the complex gel antibody/antigen by an SDS washing or an enzymatic degradation.     

High-Performance Liquid Chromatography of Copper,Mercury, Nickel,and Cadmium Bisdibenzyldithiocarbamate Complexes

Abstract

High-performance liquid chromatographic separations of Cu (II), Hg(II), Ni(II), and Cd(II) bisdibenzyldithiocarbamates at nanogram levels by adsorption chromatography on silica gel were reported. Lichrosorb Si 60 (10 um) was used as the stationary phase and benzene-cyclohexane mixtures as the mobile phase.     

A statistical comparison of silver and SYPRO Ruby staining for proteomic analysis

Silver staining has been the method most commonly employed for high sensitivity staining of proteins following two-dimensional gel electrophoresis. Whilst this method offers detection in the nanogram range it does have major drawbacks including a lack of linearity, nonstoichiometric staining of proteins, a lack of compatibility with the microchemical preparation of proteins for identification by mass spectrometric techniques, and a highly subjective assessment of the staining endpoint. SYPRO Ruby is a relatively new, ruthenium complex-based stain which is reported to offer advantages over silver, particularly in overcoming the limitations cited above. We describe a series of experiments where several protein staining procedures commonly employed are compared. To enable optimization of the in situ digestion procedure, a statistical approach has been undertaken. The effects of a variety of staining, digestion, and analysis protocols on the downstream processing of a test radiolabeled protein were studied. The data confirms that as well as offering sensitivity similar to silver, SYPRO Ruby staining is reproducible, linear, and offers a higher level of compatibility with the identification of proteins by mass spectrometry.     

Calibrated nanoinjections of mercury vapor

A simple technique for the calibration of mercury vapor chemosensors is described. It is based on a reductive deposition of a well-defined quantity of mercury (0) onto gold wire followed by thermal evaporation of the mercury into the sensor cell. The quantity of mercury on the gold wire was measured electrochemically by oxidation before and after heating, as well as after storage under different conditions. It is shown that the technique can be used for calibrated injections of nanogram quantities of mercury. The approach was applied to test the performance of ultrasensitive mercury chemoresistors.     

Solid-surface room-temperature phosphorescence detection of serotonin, tryptamine, and gramine enhanced by inorganic salts and sodium dodecyl sulfate

Room-temperature phosphorescence characteristics of serotonin (5-hydroxytryptamine), typtamine, and gramine ([3-(dimethylaminomethyl)indole]) were studied on low-background paper substrates. Several experimental parameters were optimized for maximum signal intensity. Sodium iodide, thallium(I) nitrate, silver(I) nitrate, and lead(II) nitrate were tested as phosphorescence enhancers. The effect of sodium dodecyl sulfate on the enhancement efficiency of these inorganic salts was also studied. The pH values of the solutions were adjusted to optimize the interaction between analyte molecules and solid substrates. Under the experimental conditions for maximum phosphorescence intensity, working curves ranging from two (gramine) to four (typtamine) orders of magnitude were obtained. Limits of detection at the nanogram level were estimated for the three compounds, showing the potential of solid-surface room-temperature phosphorimetry as a detection technique for biogenic amines.     

Fluoride microdetermination and its application to the analysis of rocks,soils, precipitation,and airborne dust

A method for the routine determination of nanogram amounts of fluoride is described. In a one-vessel technique, the fluoride is separated from the matrix by diffusion and then determined kinetically by its inhibiting effect on the zirconium-catalysed reaction between perborate and iodide. The method is applied to the analysis of geochemical materials, rain water, and aerosol filter samples.     

Calibrated nanoinjections of mercury vapor

A simple technique for the calibration of mercury vapor chemosensors is described. It is based on a reductive deposition of a well-defined quantity of mercury (0) onto gold wire followed by thermal evaporation of the mercury into the sensor cell. The quantity of mercury on the gold wire was measured electrochemically by oxidation before and after heating, as well as after storage under different conditions. It is shown that the technique can be used for calibrated injections of nanogram quantities of mercury. The approach was applied to test the performance of ultrasensitive mercury chemoresistors.     

Interactions of Janus Green B with double stranded DNA and the determination of DNA based on the measurement of enhanced resonance light scattering

A novel assay of DNA with a sensitivity at the nanogram level is proposed based on the measurement of enhanced resonance light scattering (RLS) signals resulting from the interaction of Janus Green B (JGB) with DNA. At pH 6.37 and ionic strength < 0.20, the RLS signals of JGB were greatly enhanced by DNA in the region of 300-650 nm characterized by three peaks at 416.0, 452.0 and 469.2 nm. The binding properties were examined using a Scatchard plot based on the measurement of the enhanced RLS data at 416.0 nm at a high JGB: DNA molar ratio (R > 2.22), and an aggregation mechanism of JGB in the presence of DNA at the nanogram level is proposed. Linear relationships can be established between the enhanced RLS intensity and DNA concentration in the range 0-3.5 micrograms ml-1 for both calf thymus DNA (ctDNA) and fish sperm DNA (fsDNA) if 2.0 x 10(-5) M JGB is employed. The limits of determination were 8.7 ng ml-1 for ctDNA and 9.9 ng ml-1 for fsDNA, respectively. Synthetic samples were analysed satisfactorily.     

Membrane-mediated ultrafast restriction digestion and subsequent rapid gel microchip electrophoresis of DNA

Ultrafast, membrane-mediated restriction digestion of DNA molecules followed by rapid gel microchip electrophoresis of the resulting fragments is described. Combination of restriction endonuclease digestion on small pore-size microfibrous membranes with sample loading and electrophoresis analysis in a multilane (up to 96) format resulted in very fast restriction digest based microscale DNA analysis. Complete digestion of several nanogram target DNA was accomplished on the microporous membrane at room temperature just in a few minutes with a single or a combination of various restriction enzymes, using only submicroliter quantities of samples and reagents. The reaction mixture containing membrane also served as sample loading device for the subsequent gel microchip electrophoresis based analysis. This work establishes methods for high-speed, high-throughput DNA analysis, featuring extremely low sample and reagent consumption, and fast restriction digestion in combination with sample loading and rapid gel microchip analysis of the resulting fragments. The entire restriction digestion, sample loading and electrophoresis analysis process required less than 20 min.