Showing papers in "Analytical Chemistry in 1996"
TL;DR: Silver staining allows a substantial shortening of sample preparation time and may, therefore, be preferable over Coomassie staining, and this work removes a major obstacle to the low-level sequence analysis of proteins separated on polyacrylamide gels.
Abstract: Proteins from silver-stained gels can be digested enzymatically and the resulting peptides analyzed and sequenced by mass spectrometry. Standard proteins yield the same peptide maps when extracted from Coomassie- and silver-stained gels, as judged by electrospray and MALDI mass spectrometry. The low nanogram range can be reached by the protocols described here, and the method is robust. A silver-stained one-dimensional gel of a fraction from yeast proteins was analyzed by nanoelectrospray tandem mass spectrometry. In the sequencing, more than 1000 amino acids were covered, resulting in no evidence of chemical modifications due to the silver staining procedure. Silver staining allows a substantial shortening of sample preparation time and may, therefore, be preferable over Coomassie staining. This work removes a major obstacle to the low-level sequence analysis of proteins separated on polyacrylamide gels.
8,437 citations
TL;DR: Improved desolvation in nanoES led to instrument-limited resolution of the signals of a glycoprotein and the ability to signal average extensively allowed the C-terminal sequencing of a 40 kDa protein.
Abstract: The nanoelectrospray ion source (nanoES) has recently been developed and described theoretically. It is different from conventional electrospray sources and from other miniaturized electrospray sources by (i) its 1−2 μm spraying orifice achieved by pulling the spraying capillary to a fine tip, (ii) its very low flow rate of ∼20 nL/min and the small size of droplets it generates, and (iii) the absence of solvent pumps and inlet valves. The fabrication and operation of nanoES needles is described in detail. Solutions with up to 0.1 M salt contents could be sprayed without sheath flow or pneumatic assist. Improved desolvation in nanoES led to instrument-limited resolution of the signals of a glycoprotein and the ability to signal average extensively allowed the C-terminal sequencing of a 40 kDa protein. Extensive mass spectrometric and tandem mass spectrometric investigation of the components of an unseparated peptide mixture was demonstrated by verification of 93% of the sequence of carbonic anhydrase. A ra...
1,786 citations
TL;DR: An analytical technique is described which combines solvent extraction with gas chromatographic (GC) analysis in a simple and inexpensive apparatus involving very little solvent consumption and is in good agreement with a convective-diffusive kinetic model.
Abstract: An analytical technique is described which combines solvent extraction with gas chromatographic (GC) analysis in a simple and inexpensive apparatus involving very little solvent consumption. A small drop (8 μL) of a water-immiscible organic solvent, containing an internal standard, is located at the end of a Teflon rod which is immersed in a stirred aqueous sample solution. After the solution has been stirred for a prescribed period of time, the probe is withdrawn from the aqueous solution, and the organic phase is sampled with a microsyringe and injected into the GC for quantification. The observed rate of solvent extraction is in good agreement with a convective−diffusive kinetic model. Analytically, the relative standard deviation of the method is 1.7% for a 5.00-min extraction of the analyte 4-methylacetophenone into n-octane.
1,191 citations
TL;DR: In this paper, the authors describe a new technique for sample preparation, accelerated solvent extraction (ASE), that combines elevated temperatures and pressures with liquid solvents, and investigate the effects of various operational parameters (i.e., temperature, pressure, and volume of solvent used) on the performance of ASE.
Abstract: We describe a new technique for sample preparation, accelerated solvent extraction (ASE), that combines elevated temperatures and pressures with liquid solvents. The effects of various operational parameters (i.e., temperature, pressure, and volume of solvent used) on the performance of ASE were investigated. The solvents used are those normally used for standard liquid extraction techniques like Soxhlet or sonication. We found the recoveries of polycyclic aromatic hydrocarbons, polychlorinated biphenyls, and total petroleum hydrocarbons from reference materials using ASE to be quantitative. The extraction time for 1−30-g samples is less than 15 min, and the volume of solvent is 1.2−1.5 times that of the extraction cell containing the sample. No evidence was seen for thermal degradation during the extraction of temperature-sensitive compounds.
1,109 citations
TL;DR: The feasibility of performing high-speed DNA analyses in microfabricated integrated fluidic systems is established, demonstrating that challenging amplifications of diagnostically interesting targets can also be performed.
Abstract: Microfabricated silicon PCR reactors and glass capillary electrophoresis (CE) chips have been successfully coupled to form an integrated DNA analysis system. This construct combines the rapid thermal cycling capabilities of microfabricated PCR devices (10 °C/s heating, 2.5 °C/s cooling) with the high-speed (<120 s) DNA separations provided by microfabricated CE chips. The PCR chamber and the CE chip were directly linked through a photolithographically fabricated channel filled with hydroxyethylcellulose sieving matrix. Electrophoretic injection directly from the PCR chamber through the cross injection channel was used as an “electrophoretic valve” to couple the PCR and CE devices on-chip. To demonstrate the functionality of this system, a 15 min PCR amplification of a β-globin target cloned in M13 was immediately followed by high-speed CE chip separation in under 120 s, providing a rapid PCR−CE analysis in under 20 min. A rapid assay for genomic Salmonella DNA was performed in under 45 min, demonstrating ...
870 citations
TL;DR: The performance of the silica rods was much better at a high flow rate than that of conventional columns packed with 5 μm C(18) silica particles having 12 and 30 nm pores, especially for high molecular weight species.
Abstract: Continuous porous silica rods consisting of a mesoporous (pore size, 14 or 25 nm) silica skeleton of ∼1 μm size and through-pores of ∼1.7 μm were prepared and derivatized to C18 phase by on-column reaction with octadecyldimethyl-(N,N-diethylamino)silane. The C18 silica rods gave plate heights of 10−20 μm for aromatic hydrocarbons in 80% methanol and 20−40 μm for insulin in acetonitrile−water mixtures in the presence of trifluoroacetic acid. The performance of the silica rods was much better at a high flow rate than that of conventional columns packed with 5 μm C18 silica particles having 12 and 30 nm pores, especially for high molecular weight species.
856 citations
TL;DR: It is concluded that the elimination of uninformative variables can improve predictive ability and be evaluated on simulated data.
Abstract: A new method for the elimination of uninformative variables in multivariate data sets is proposed. To achieve this, artificial (noise) variables are added and a closed form of the PLS or PCR model is obtained for the data set containing the experimental and the artificial variables. The experimental variables that do not have more importance than the artificial variables, as judged from a criterion based on the b coefficients, are eliminated. The performance of the method is evaluated on simulated data. Practical aspects are discussed on experimentally obtained near-IR data sets. It is concluded that the elimination of uninformative variables can improve predictive ability.
843 citations
TL;DR: This unique microextraction system is simple and flexible, permits automated backwashing, consumes only microquantities of organic solvents, and is capable of being coupled with other analytical systems.
Abstract: An organic microdrop (∼1.3 μL) is suspended inside a flowing aqueous drop from which the analyte is extracted. The drop-in-drop system is achieved by a multitube assembly. The aqueous phase is continuously delivered to the outer drop and is aspirated away from the bottom meniscus of the drop. After the sampling/extraction period, a wash solution replaces the sample/reagent in the aqueous layer, resulting in a clear outer aqueous drop housing a colored organic drop containing the extracted material. This also results in an automatic backwash. The color intensity of the organic drop, related to the analyte concentration, is monitored by a light-emitting diode based absorbance detector. After the analytical cycle, the organic drop is removed and replaced by a new one. The performance of the system is illustrated with the determination of sodium dodecyl sulfate (a methylene blue active substance) extracted as an ion pair into chloroform. This unique microextraction system is simple and flexible, permits autom...
697 citations
TL;DR: In this article, various surface modification procedures were used on glassy carbon (GC) electrodes to yield surfaces with low oxide content or which lack specific oxide functional groups, and monolayers of several different adsorbates were formed on GC surfaces before electrochemical evaluation.
Abstract: Various well-established and novel surface modification procedures were used on glassy carbon (GC) electrodes to yield surfaces with low oxide content or which lack specific oxide functional groups. In addition, monolayers of several different adsorbates were formed on GC surfaces before electrochemical evaluation. Both the nonspecific monolayer adsorbates and reagents which chemisorb to specific functional groups were observed on the surface with Raman and photoelectron spectroscopy. The various GC surfaces were then evaluated for their electron transfer reactivity with nine redox systems in aqueous electrolyte, including Ru(NH3)62+/3+, Fe(CN6)3-/4-, ascorbic acid, and Feaq3+/2+. The nine systems were categorized according to their kinetic sensitivity to surface modification. Several, including Ru(NH3)62+/3+, are insensitive to surface modifications and are considered outer sphere. Feaq3+/2+, Vaq2+/3+, and Euaq2+/3+ are catalyzed by surface carbonyl groups and are very sensitive to the removal of surface...
669 citations
TL;DR: Surface plasmon resonance studies showed that His-tagged proteins adsorbed on the NTA-SAM retained a greater ability to participate in binding interactions with proteins in solution than protein immobilized in a thin dextran gel layer by covalent coupling.
Abstract: This paper reports the generation of a self-assembled monolayer (SAM) that selectively binds proteins whose primary sequence terminates with a His-tag: a stretch of six histidines commonly incorporated in recombinant proteins to simplify purification. The SAM was prepared by the adsorption onto a gold surface of a mixture of two alkanethiols: one thiol that terminated with a nitrilotriacetic acid (NTA) group, a group that forms a tetravalent chelate with Ni(II), and a second thiol that terminated with a tri(ethylene glycol) group, a group that resists protein adsorption. His-tagged proteins bound to the SAM by interaction of the histidines with the two vacant sites on Ni(II) ions chelated to the surface NTA groups. Studies with model proteins showed the binding was specific for His-tagged proteins and required the presence of Ni(II) on the surface. Immobilized His-tagged proteins were kinetically stable in buffered saline at pH 7.2 but could be desorbed by treatment with 200 mM imidazole. Surface plasmo...
597 citations
TL;DR: A new optical sensor based on covalent immobilization of a newly synthesized calcium-selective, long-wavelength, fluorescent indicator has been constructed, with a response dynamic range optimal for physiological measurements.
Abstract: A new optical sensor based on covalent immobilization of a newly synthesized calcium-selective, long-wavelength, fluorescent indicator has been constructed, with a response dynamic range optimal for physiological measurements. Immobilization occurs via photoinitiated copolymerization of the indicator with acrylamide on the distal end of a silanized 125 μm diameter multimode optical fiber. The working lifetime of this sensor is limited only by photobleaching of the indicator. Due to the inherent hydrophilic nature of the acrylamide polymer, the response time of this new sensor is governed by simple aqueous diffusion of the ionic calcium. This results in sensor response times fast enough to monitor some concentration fluctuations at physiological rates. The ability to monitor calcium concentration fluctuations in a high background level of magnesium is also demonstrated with a calculated selectivity of 10-4.5.
TL;DR: Results indicate that there is a reverse-phase chromatographic-like dimension in the sample-matrix preparation procedures that can be exploited to optimize the analysis of MALDI mass spectra.
Abstract: Sample−matrix preparation procedures are shown to greatly influence the quality of the matrix-assisted laser desorption/ionization (MALDI) mass spectra of peptides and proteins. In particular, dramatic mass discrimination effects are observed when the matrix 4-hydroxy-α-cyanocinnamic acid is used for analyzing complex mixtures of peptides and proteins. The discrimination effects are found to be strongly dependent on the sample−matrix solution composition, pH, and the rates at which the sample−matrix cocrystals are grown. These findings demonstrate the need to exercise great care in performing and interpreting the MALDI analysis of biological samples. The results also indicate that there is a reverse-phase chromatographic-like dimension in the sample−matrix preparation procedures that can be exploited to optimize the analysis. The present work describes the conditions under which the majority of components of a complex mixture of peptides and proteins can be successfully measured.
TL;DR: Doing a thorough method validation can be tedious, but the consequences of not doing it right are wasted time, money, and resources as mentioned in this paper, which is wasted time and money.
Abstract: Doing a thorough method validation can be tedious, but the consequences of not doing it right are wasted time, money, and resources
TL;DR: A suspension polymerization technique suitable for molecular imprinting is described, based on the use of a liquid perfluorocarbon as the dispersing phase, which produces polymer beads, with almost quantitative yield, which can be used after only a simple washing step.
Abstract: A suspension polymerization technique suitable for molecular imprinting is described, based on the use of a liquid perfluorocarbon as the dispersing phase. This dispersant does not interfere with the interactions between functional monomers and print molecules required for the recognition process during molecular imprinting. The method produces polymer beads, with almost quantitative yield, which can be used after only a simple washing step. An acrylate polymer with perfluorocarbon and poly(oxyethylene) ester groups was used to stabilize an emulsion of functional monomer, cross-linker, print molecule, initiator, and porogenic solvent in perfluoro(methylcyclohexane). Initiation of polymerization by UV irradiation resulted in polymer beads. The average bead size could be controlled between about 50 and 5 μm by varying the amount of stabilizing polymer. SEM of the beads indicated spherical particles with morphology typical of beads made by suspension polymerization. The technique was applicable to a range of...
TL;DR: A novel and useful new chromatography system in which surface properties and the resulting function of the HPLC stationary phase are controlled by externalTemperature changes should be effective in biological and biomedical separations of peptides and proteins using only aqueous mobile phases.
Abstract: A new concept in chromatography is proposed that utilizes a temperature-responsive surface with a constant aqueous mobile phase. The surface of the silica stationary phase in high-performance liquid chromatography (HPLC) has been modified with temperature-responsive polymers to exhibit temperature-controlled hydrophilic/hydrophobic changes. Poly(N-isopropylacrylamide) (PIPAAm) was grafted onto (aminopropyl)silica using an activated ester-amine coupling method. These grafted silica surfaces show hydrophilic properties at lower temperatures which, as temperature increases, transform to hydrophobic surface properties. The elution profile of five mixed steroids on an HPLC column packed with this material depends largely on the temperature of the aqueous mobile phase. Retention times increase with increasing temperature without any change in the eluent. Changes in the retention times of hydrophobic steroids were larger than those for hydrophilic steroids. The temperature-responsive interaction between PIPAAm-modified silica and these steroids is proposed to result from changes in the surface properties of the HPLC stationary phase by the transition of hydrophilic/hydrophobic surface-grafted IPAAm polymers. We demonstrate a novel and useful new chromatography system in which surface properties and the resulting function of the HPLC stationary phase are controlled by external temperature changes. This method should be effective in biological and biomedical separations of peptides and proteins using only aqueous mobile phases.
TL;DR: A new amperometric microsensor for detection of dissolved hydrogen sulfide (H2S) in aquatic environments was developed in this paper, which is based on the same principle as the Clark-type oxygen microsensors.
Abstract: A new amperometric microsensor for detection of dissolved H2S in aquatic environments was developed The design of the microsensor is based on the same principle as the Clark-type oxygen microsensor The sensor is equipped with a glass-coated platinum working electrode and a platinum guard electrode positioned in an outer glass casing (tip diameter 20−100 μm) Both working electrode and guard electrode were polarized at a fixed value in the range from +85 to + 150 mV with respect to a counter electrode The outer casing is sealed with a thin silicone membrane and filled with a buffered electrolyte solution containing ferricyanide (K3[Fe(CN)6]) as redox mediator Hydrogen sulfide penetrates the silicone membrane and is oxidized by K3[Fe(CN)6], resulting in the formation of elemental sulfur and ferrocyanide (K4[Fe(CN)6]) The latter is electrochemically reoxidized at the exposed end of the platinum working electrode, thereby creating a current that is directly proportional to the dissolved H2S concentration
TL;DR: Based on preliminary results, the PCGC-based approach holds promise for accurately determining (14)C ages on compounds specific to a given source within complex, heterogeneous samples.
Abstract: This paper describes the application of a novel, practical approach for isolation of individual compounds from complex organic matrices for natural abundance radiocarbon measurement. This is achieved through the use of automated preparative capillary gas chromatography (PCGC) to separate and recover sufficient quantities of individual target compounds for 14C analysis by accelerator mass spectrometry (AMS). We developed and tested this approach using a suite of samples (plant lipids, petroleums) whose ages spanned the 14C time scale and which contained a variety of compound types (fatty acids, sterols, hydrocarbons). Comparison of individual compound and bulk radiocarbon signatures for the isotopically homogeneous samples studied revealed that Δ14C values generally agreed well (±10%). Background contamination was assessed at each stage of the isolation procedure, and incomplete solvent removal prior to combustion was the only significant source of additional carbon. Isotope fractionation was addressed thr...
TL;DR: An integrated monolithic device that performs an automated biochemical procedure is demonstrated that mixes a DNA sample with a restriction enzyme in a 0.7-nL reaction chamber and injects the fragments onto a 67-mm-long capillary electrophoresis channel for sizing.
Abstract: An integrated monolithic device (8 mm × 10 mm) that performs an automated biochemical procedure is demonstrated. The device mixes a DNA sample with a restriction enzyme in a 0.7-nL reaction chamber and after a digestion period injects the fragments onto a 67-mm-long capillary electrophoresis channel for sizing. Materials are precisely manipulated under computer control within the channel structure using electrokinetic transport. Digestion of the plasmid pBR322 by the enzyme HinfI and fragment analysis are completed in 5 min using 30 amol of DNA and 2.8 × 10-3 unit of enzyme per run.
TL;DR: The resonance light-scattering technique, using a spectrofluorometer, was first developed as a sensitive instrumental analysis method as mentioned in this paper, and linear relationships between the enhanced extents of lightscattering and the concentrations of nucleic acids in the range of 1.8 × 10-7−10.
Abstract: The resonance light-scattering technique, using a spectrofluorometer, was first developed as a sensitive instrumental analysis method. At pH 7.48 and ionic strength 0.004, the extent of light-scattering of α,β,γ,δ-tetrakis[4-(trimethylammoniumyl)phenyl]porphine (TAPP) is enhanced by nucleic acids near 432 nm. There are linear relationships between the enhanced extents of light-scattering and the concentrations of nucleic acids in the range of 1.8 × 10-7−10.8 × 10-7 M for calf thymus and fish sperm DNA and in the range of 1.8 × 10-7−1.8 × 10-6 M for yeast RNA. The limit of determination (3σ) is 4.1 × 10-8 M for calf thymus DNA, 4.6 × 10-8 M for fish sperm DNA, and 6.7 × 10-8 M for yeast RNA. Mechanism study indicates that nucleic acids react with the title porphyrin in two modes, depending on the concentrations of nucleic acids. When the molar ratio of nucleic acids to TAPP is smaller than 4:1, the hypochromicity and fluorescence quenching of TAPP by nucleic acids appear, and the enhancement of resonance l...
TL;DR: An immunoassay performed using a microchip electrophoretic system achieves the determination of cortisol in blood serum over the range of clinical interest (1-60 micrograms/dL) without the need for extraction or other sample preparation steps.
Abstract: An immunoassay performed using a microchip electrophoretic system is described. Separation and quantitation of free and bound labeled antigen in a competitive assay are carried out in channels micromachined into fused silica substrates. Such microchips are attractive because of their small size, ruggedness, and amenability to automated handling. The assay achieves the determination of cortisol in blood serum over the range of clinical interest (1−60 μg/dL) without the need for extraction or other sample preparation steps. The separation is performed in less than 30 s. Very high throughput is possible by operating the assay in multiple channels in parallel. These characteristics make microchip electrophoretic systems a promising technology for the rapid analysis of clinical samples.
TL;DR: The results show that the contacting conductive liquid, in effect, enlarges the electrode area on the liquid side and thereby changes the parallel resonant frequency, and by proper design of the QCM measurement, perturbing effects due to the liquid's electrical properties can be circumvented.
Abstract: Recently, several reports have shown that when one side of a quartz crystal microbalance (QCM) is exposed to a liquid, the parallel (but not the series) resonant frequency is influenced by the conductivity and dielectric constant of the liquid. The effect is still controversial and constitutes a serious complication in many applications of the QCM in liquid environments. One suggestion has been that acoustically induced surface charges couple to charged species in the conducting liquid. To explore this effect, we have measured the parallel and the series mode resonance frequencies, and the corresponding Q factors, for a QCM with one side facing a liquid. These four quantities have all been measured versus liquid conductivity, using a recently developed experimental setup. It allows the simultaneous measurement of the resonant frequency and the Q factor of an oscillating quartz crystal, intermittently disconnected from the driving circuit. Based on these results, a simple model together with an equivalent circuit for a quartz crystal exposed to a liquid is presented. The analysis shows that it is not necessary to infer the existence of surface charges (or other microscopic phenomena such as electrical double layers) to account for the influence of the liquid's electrical properties on the resonant frequency. Our results show that the contacting conductive liquid, in effect, enlarges the electrode area on the liquid side and thereby changes the parallel resonant frequency. By proper design of the QCM measurement, perturbing effects due to the liquid's electrical properties can be circumvented.
TL;DR: The proposed sensor exhibits very good selectivity for Zn(2+) over other cations and can be used in a wide pH range and has also been possible to use this assembly as an indicator electrode in potentiometric titrations involving zinc ions.
Abstract: A PVC-based membrane of 2,2,2-cryptand exhibits a very good response for Zn2+ in a wide concentration range (from 2.06 ppm to 6.54 × 103 ppm) with a slope of 22.0 mV/decade of Zn2+ concentration. The response time of the sensor is <10 s, and the membrane can be used for more than 3 months without any observed divergence in potentials. The proposed sensor exhibits very good selectivity for Zn2+ over other cations and can be used in a wide pH range (2.8−7.0). It has also been possible to use this assembly as an indicator electrode in potentiometric titrations involving zinc ions.
TL;DR: The reactivity and packing density of the MUA-NHSS esters are investigated in detail by reacting these intermediates with ammonia (NH3), and nearly all of theMUA molecules (∼80%) are converted to amides.
Abstract: A monolayer of poly(l-lysine) (PL) is attached covalently via amide bonds to an alkanethiol self-assembled monolayer (SAM) on a gold surface. The amide bond is formed in two steps: the terminal carboxylic acid groups of an 11-mercaptoundecanoic acid (MUA) SAM are first activated to the N-hydroxysulfosuccinimide (NHSS) ester, followed by reaction of this MUA-NHSS ester monolayer with the amino groups of PL to create multiple amide bond linkages to the surface. The reactivity and packing density of the MUA-NHSS esters are investigated in detail by reacting these intermediates with ammonia (NH3). In the NH3 experiments, approximately 50% of the carboxylic acids in the MUA monolayer are converted to amides during the first cycle of this two-step surface reaction. This reaction yield of 50% is limited by the steric packing of the NHSS ester intermediate. However, after three cycles of MUA activation to the NHSS ester and reaction with NH3, nearly all of the MUA molecules (∼80%) are converted to amides. Polari...
TL;DR: The results show that charge-charge interactions not only lower the activation energy for dissociation but also can dramatically change the fragmentation, most likely through changes in the gas-phase conformation of the ion.
Abstract: A new method for the dissociation of large ions formed by electrospray ionization is demonstrated. Ions trapped in a Fourier transform mass spectrometer at pressures below 10-8 Torr are dissociated by elevating the vacuum chamber to temperatures up to 215 °C. Rate constants for dissociation are measured and found to be independent of pressure below 10-7 Torr. This indicates that the ions are activated by absorption of blackbody radiation emitted from the chamber walls. Dissociation efficiencies as high as 100% are obtained. There is no apparent mass limit to this method; ions as large as ubiquitin (8.6 kDa) are readily dissociated. Thermally stable ions, such as melittin 3+ (2.8 kDa), did not dissociate at temperatures up to 200 °C. This method is highly selective for low-energy fragmentation, from which limited sequence information can be obtained. From the temperature dependence of the dissociation rate constants, Arrhenius activation energies in the low-pressure limit are obtained. The lowest energy di...
TL;DR: The toluene-based assay showed excellent enantioselectivity, the cross-reactivity of the R enantiomer being only 1%, which is better than that demonstrated by biological antibodies, and the aqueous buffer based assay showed high substrate selectivity for propranolol in the presence of structurally similar β-blockers.
Abstract: Antibody mimics have been prepared by molecular imprinting of (S)-propranolol and applied in the development of radioligand binding assays for the imprint species. In the assays, polymer particles, radioligand, and analyte were incubated either in an organic solvent or an aqueous buffer, with similarly high sensitivity under both set of conditions. Optimization of the assay conditions led to 100-1000-fold improvements in the limit of determination and 100-1000-fold reductions of the amount of imprinted polymer used, compared with previous studies of this novel type of assay. The present assay uses only 10-50 μg of polymer, and the limit of determination is about 6 nM. The toluene-based assay showed excellent enantioselectivity, the cross-reactivity of the R enantiomer being only 1%, which is better than that demonstrated by biological antibodies. The aqueous buffer based assay showed high substrate selectivity for propranolol in the presence of structurally similar β-blockers. The different selectivity profiles obtained are due to a different balance between hydrophobic and polar interactions in toluene and water, since polar interactions, such as hydrogen bonds, are strong in apolar solvents and hydrophobic interactions are strong in water.
TL;DR: Methods for identifying and sequencing phosphopeptides in simple mixtures, such as HPLC fractions, at the subpicomole level by (+) ion matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-MS) are presented.
Abstract: In this paper we present methods for identifying and sequencing phosphopeptides in simple mixtures, such as HPLC fractions, at the subpicomole level by (+) ion matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-MS). Data are presented which indicate that when a reflectron time-of-flight mass spectrometer is used, MALDI can distinguish tyrosine phosphorylation from serine and threonine phosphorylation for peptides containing a single phosphate group. Phosphopeptides are identified in the (+) ion MALDI reflector spectrum by the presence of [MH − H3PO4]+ and [MH − HPO3]+ fragment ions formed by metastable decomposition. An abundant [MH − H3PO4]+ ion, accompanied by a weaker [MH − HPO3]+ ion indicates that the peptide is most likely phosphorylated on serine or threonine. In contrast, phosphotyrosine-containing peptides generally exhibit [MH − HPO3]+ fragment ions and little, if any [MH − H3PO4]+. Ambiguities do arise, most often with phosphopeptides that contain residues which readily lo...
TL;DR: Problems inherent to the conventional mobility determination method such as a variable electroosmotic flow during the run and migration through unthermostated regions of the capillary are eliminated with the use of the proposed method.
Abstract: A new method for accurately determining effective mobilities and electroosmotic flow rates for capillary electrophoresis is described. The proposed method can be performed using most commercial capillary electrophoresis instruments. Problems inherent to the conventional mobility determination method such as a variable electroosmotic flow during the run and migration through unthermostated regions of the capillary are eliminated with the use of the proposed method. In addition, very low effective mobilities and electroosmotic flow rates can be measured quickly and reproducibly. Also, cation mobilities and anion mobilities can be measured in a single run regardless of the magnitude or direction of the electroosmotic flow.
TL;DR: With this approach, dopamine release elicited by two electrical stimulus pulses at 10 Hz was detected with fastscan cyclic voltammetry in vivo and high-pass filtering discriminated against ascorbic acid, DOPAC, and acidic pH changes, three common interferences in vivo.
Abstract: Many of the molecules involved in biological signaling processes are easily oxidized and have been monitored by electrochemical methods. Temporal response, spatial considerations, and sensitivity of the electrodes must be optimized for the specific biological application. To monitor exocytosis from single cells in culture, constant potential amperometry offers the best temporal resolution, and a low-noise picoammeter improves the detection limits. Smaller electrodes, with 1-μm diameters, provided spatial resolution sufficient to identify the locations of release sites on the surface of single cells. For the study of neurotransmitter release in vivo, larger cylindrical microelectrodes are advantageous because the secreted molecules come from multiple terminals near the electrode, and the greater amounts lead to a larger signal that emerges from the Johnson noise of the current amplifier. With this approach, dopamine release elicited by two electrical stimulus pulses at 10 Hz was detected with fast-scan cyc...
TL;DR: A miniaturized device for simultaneous measurement of glucose and lactate levels was produced by means of photopatterning of enzyme-containing photosensitive membrane precursors and subcutaneous measurements of glucose levels in pigs were close to the corresponding blood levels obtained without in vivo calibration.
Abstract: A miniaturized device for simultaneous measurement of glucose and lactate levels was produced by means of photopatterning of enzyme-containing photosensitive membrane precursors. This device shows no cross-talk and a lifetime for both the glucose and the lactate sensors of more than 2 weeks when continuously operated in undiluted bovine serum. Linear response ranges of up to 40 mM for glucose and 25 mM for l-lactate, in combination with 95% response times of 0.98 with respect to laboratory techniques. Subcutaneous measurements of glucose levels in pigs were close to the corresponding blood levels obtained without in vivo calibration.
TL;DR: An electrochemical biosensor for the detection of short DNA sequences related to the human immunodeficiency virus type 1 (HIV-1) and the direct adsorptive chronopotentiometric stripping measurements of trace levels of various HIV-1 DNAs are reported.
Abstract: An electrochemical biosensor for the detection of short DNA sequences related to the human immunodeficiency virus type 1 (HIV-1) is described. The sensor relies on the immobilization and hybridization of the 21- or 42-mer single-stranded oligonucleotide from the HIV-1 U5 long terminal repeat (LTR) sequence at carbon paste or strip electrodes. The extent of hybridization between the complementary sequences is determined by the enhancement of the chronopotentiometric peak of the Co(phen)33+ indicator. Numerous factors affecting the probe immobilization, target hybridization, and indicator binding reactions are optimized to maximize the sensitivity and speed the assay time. A detection limit of 4 × 10-9 M HIV-1 U5 LTR segment is reported following a 30 min hybridization. The hybridization biosensor format obviates the use of radioisotopes common in radioactive methods for the detection of HIV-1 DNA. We also report on the direct adsorptive chronopotentiometric stripping measurements of trace levels of various...