Sequential double fluorescent detections of total proteins and phosphoproteins in SDS‐PAGE

A fluorescent staining technique, using selective chelation with fluorophore and metal ion to the phosphate groups of phosphoproteins in SDS‐PAGE is described. As a fluorescent dye and a metal ion, Fura 2 pentapotassium salt and Al³⁺ were employed, respectively. The staining method, Fura 2 stain, has sensitivities of 16–32 ng of α‐casein and β‐casein, 62 ng of ovalbumin, phosvitin, and κ‐casein using an ultraviolet transilluminator. Furthermore, Fura 2 stain is able to carry out continuative double detection of total proteins and phosphoproteins on the same gel within 3.5 h. Consequently, selective phosphoprotein and total protein detections could be obtained without other poststaining. Considering the low cost, simplicity, and speed, Fura 2 staining may provide great practicalities in routine phosphoproteomics research.     

A rapid and simple 8‐quinolinol‐based fluorescent stain of phosphoproteins in polyacrylamide gel after electrophoresis

In order to obtain an easy and rapid protocol to visualize phosphoproteins in SDS‐PAGE, a fluorescent detection method named 8‐Quinolinol (8‐Q) stain is described. 8‐Q can form ternary complexes in the gel matrix contributed by the affinity of aluminum ion (Al³⁺) to the phosphate groups on the proteins and the metal chelating property of 8‐Quinolinol, exhibiting strong fluorescence in ultraviolet light. It can visualize as little as 4～8 ng of α‐casein and β‐casein, 16～32 ng of ovalbumin and κ‐casein which is more sensitive than Stains‐All but less sensitive than Pro‐Q Diamond. The protocol of 8‐Q requires only 70 min in 0.75 mm mini‐size or 1.0 mm large‐size gels with five changes of solutions without destaining step; Pro‐Q takes at least 250 min with 11 changes of solutions. In addition, the new method was confirmed by the study of dephosphorylation and LC‐MS/MS, respectively. The approach to visualize phosphoprotein utilizing 8‐Q could be an alternative to simplify the analytical operations for phosphoproteomics research.     

Solid‐phase extraction of plant thionins employing aluminum silicate based extraction columns

Thionins belong to a family of cysteine‐rich, low‐molecular‐weight (～5 KDa) biologically active proteins in the plant kingdom. They display a broad cellular toxicity against a wide range of organisms and eukaryotic cell lines. Thionins protect plants against different pathogens, including bacteria and fungi. A highly selective solid‐phase extraction method for plant thionins is reported deploying aluminum silicate (3:2 mullite) powder as a sorbent in extraction columns. Mullite was shown to considerably improve selectivity compared to a previously described zirconium silicate embedded poly(styrene‐co‐divinylbenzene) monolithic polymer. Due to the presence of aluminum(III), mullite offers electrostatic interactions for the selective isolation of cysteine‐rich proteins. In comparison to zirconium(IV) silicate, aluminum(III) silicate showed reduced interactions towards proteins which resulted into superior washings of unspecific compounds while still retaining cysteine‐rich thionins. In the presented study, European mistletoe, wheat and barley samples were subjected to solid‐phase extraction analysis for isolation of viscotoxins, purothionins and hordothionins, respectively. Matrix‐assisted laser desorption/ionization time of flight mass spectroscopy was used for determining the selectivity of the sorbent toward thionins. The selectively retained thionins were quantified by colorimetric detection using the bicinchoninic acid assay. For peptide mass‐fingerprint analysis tryptic digests of eluates were examined.     

Simultaneous capillary electrophoretic analysis of inorganic anions and cations in post‐blast extracts of acid–aluminum mixtures

Bursts resulting from the chemical reaction between hydrochloric or nitric acid with aluminum foils are very often committed by the young delinquency in western countries because of its easiness of achievement. A fast, simple, selective, and cost‐effective method allowing the simultaneous detection of chloride and nitrate anions and aluminum(III) was thus required. This article focused on the development and validation of a CE method using a BGE containing 2,6‐pyridinedicarboxylic acid (PDC) acting as both an anionic chromophore and as an aluminum(III) complexing agent. First, the achievement of the speciation diagram of Al(III) in the presence of PDC allowed the choice of pH conditions for which aluminum(III) was globally anionic. The study of the selectivity for Al(III) in the presence of ten other cationic species potentially present in post‐blast residues dictated the choice of the PDC concentration at 20 mM. The validation step next demonstrated the figures of merit of the method, with an intermediate precision for Al(III) of 2% on normalized migration times and 3.5% on corrected areas. Finally, this method was used for analyses of real post‐blast extracts from acid–aluminum mixtures.     

The characteristic of photoluminescence of tris‐(7‐substituted‐8‐hydroxyquinoline) aluminum complexes and polymeric complexes

The 7‐allyl‐ and 7‐(2‐methylvinyl)‐functionalized derivatives of 8‐hydroquinoline are synthesized by Claisen rearrangement and double bond rearrangement respectively. Then 7‐allyl‐8‐hydroquinoline (C) and 7‐(2‐methylvinyl)‐8‐hydroquinoline (D) are reacted with aluminum chloride to afford the corresponding tris‐(7‐allyl‐8‐hydroxyquinoline) aluminum complex (F) and tris‐(7‐(2‐methylvinyl)‐8‐hydroxyquinoline) aluminum complex (G). The photoluminescence of complex (F) or (G), compared with that of tris‐(8‐hydroxyquinoline) aluminum complex (E), all showed a red shift in emission wavelengths in different solvents, such as chloroform, hexane and ethanol. For two substituents containing an external double bond, the 2‐methylvinyl group gives a larger red shift in the emission wavelength than the allyl group. The X‐ray crystal structure indicates that 7‐(2‐methylvinyl)‐8‐hydroxyquinoline (D) is a trans‐isomer. The styrene and 7‐allyl‐8‐hydroxyquinoline copolymer, and the styrene and 7‐(2‐methylvinyl)‐8‐hydroxyquinoline copolymer are also reported. Further reactions of the copolymer are then performed by adding aluminum(III) chloride and ligands 8‐hydroxyquinoline. The spectroscopic characteristics of these aluminum(III) polymeric complexes are discussed. Copyright © 2003 John Wiley & Sons, Ltd.     

Highly selective enrichment of phosphopeptides using poly(dibenzo‐18‐crown‐6) as a solid‐phase extraction material

A poly(dibenzo‐18‐crown‐6) was used as a new solid‐phase extraction material for the selective enrichment of phosphopeptides. Isolation of phosphopeptides was achieved based on specific ionic interactions between poly(dibenzo‐18‐crown‐6) and the phosphate group of phosphopeptides. Thus, a method was developed and optimized, including loading, washing and elution steps, for the selective enrichment of phosphopeptides. To assess this potential, tryptic digest of three proteins (α‐ casein, β‐casein and ovalbumin) was applied on poly(dibenzo‐18‐crown‐6). The nonspecific products were removed by centrifugation and washing. The spectrometric analysis was performed using matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry. Highly selective enrichment of both mono‐ and multiphosphorylated peptides was achieved using poly(dibenzo‐18‐crown‐6) as solid‐phase extraction material with minimum interference from nonspecific compounds. Furthermore, evaluation of the efficiency of the poly(dibenzo‐18‐crown‐6) was performed by applying the digest of egg white. Finally, quantum mechanical calculations were performed to calculate the binding energies to predict the affinity between poly(dibenzo‐18‐crown‐6) and various ligands. The newly identified solid‐phase extraction material was found to be a highly efficient tool for phosphopeptide recovery from tryptic digest of proteins.     

In‐tube solid‐phase microextraction capillary column packed with mesoporous TiO2 nanoparticles for phosphopeptide analysis

In this study, an in‐tube solid‐phase microextraction column packed with mesoporous TiO₂ nanoparticles, coupled with MALDI–TOF–MS, was applied to the selective enrichment and detection of phosphopeptides in complex biological samples. The mesoporous TiO₂ nanoparticles with high specific surface areas, prepared by a sol–gel and solvothermal method, were injected into the capillary using a slurry packing method with in situ polymerized monolithic segments as frits. Compared with the traditional solid‐phase extraction method, the TiO₂‐packed column with an effective length of 1 cm exhibited excellent selectivity (α‐casein/β‐casein/BSA molar ratio of 1:1:100) and sensitivity (10 fmol of a β‐casein enzymatic hydrolysis sample) for the enrichment of phosphopeptides. These performance characteristics make this system suitable for the detection of phosphorylated peptides in practical biosamples, such as nonfat milk.     

Dual‐column capillary microextraction (CME) combined with electrothermal vaporization inductively coupled plasma mass spectrometry (ETV‐ICP‐MS) for the speciation of arsenic in human hair extracts

In this work, dual‐column capillary microextraction (CME) system consisting of N‐(2‐aminoethyl)‐3‐aminopropyltrimethoxysilane (AAPTS)‐silica coated capillary (C1) and 3‐mercaptopropyl trimethoxysilane (MPTS)‐silica coated capillary (C2) was developed for sequential separation/preconcentration of arsenite [As(III)], arsenate [As(V)], monomethylarsonic acid [MMA(V)] and dimethylarsinic acid [DMA(V)] in the extracts of human hair followed by electrothermal vaporization inductively coupled plasma mass spectrometry (ETV‐ICP‐MS) detection with iridium as permanent modifier. Various experimental parameters affecting the dual‐column microextraction of different As species had been investigated in detail. It was found that at pH 9, As(V) and MMA could be quantitatively retained by C1 and only As(III) could be quantitatively retained by C2. With the aid of valve switching, As(V)/MMA(V) retained on C1 and As(III) retained on C2 could be sequentially desorbed by 10 µl of 0.01 mol l^?1 HNO₃ [for As(V)], 0.1 mol l^?1 HNO₃ [for MMA(V)] and 0.2 mol l^?1 HNO₃‐3% thiourea (m/v) [for As(III)], respectively, the eluents were immediately introduced into the Ir‐coated graphite tubes for further ETV‐ICP‐MS detection. With two‐step ETV pyrolysis program, Cl^? in the sample matrix could be in situ removed, and the total As in the human hair extracts or digested solution could be interference‐free, determined by ETV‐ICP‐MS. DMA(V) in the human hair extracts was obtained by subtraction of total As in the human hair extracts from other three As species. Under the optimized conditions, the detection limits (3 σ) of the method were 3.9 pg ml^?1 for As(III), 2.7 pg ml^?1 for As(V), 2.6 pg ml^?1 for MMA(V) and 124 pg ml^?1 for total As with the relative standard deviations less than 7.0% (C = 0.1 ng ml^?1, n = 7), and the enrichment factor was 286, 262 and 260 for As(III), As(V) and MMA(V), respectively. The developed method was successfully applied for the speciation of arsenic in the extracts of human hair. Copyright © 2009 John Wiley & Sons, Ltd.     

Indirect Atomic Absorption Determination of Cocaine via Formation and Liquid‐liquid Extraction of the Iron(III)‐cocaine Complex

The principles of metal‐alkaloid ion‐pair formation and liquid‐liquid extraction are applied to the development of a sensitive and convenient atomic absorption spectrophotometery (AAS) method for the indirect determination of cocaine. In an aqueous medium of 5 M hydrochloric acid, cocaine is protonated and is associated with tetrachloro ferrate (III) anion prior to its extraction into 1,2‐dichloroethane. The critical experimental variables were identified and optimized. The method is simple and reproducible with a detection limit (DL) of 0.1 ng cm^?3 cocaine in water, a relative standard deviation of 0.07 (n = 12), and the calibration graph was linear up to 50 ng cm^?3 cocaine.     

Highly Selective Molecularly Imprinted Polymer Sensor for Indium Detection Based on Recognition of In‐Alizarin Complexes

A highly selective molecularly imprinted polymer electrochemical sensor for In³⁺ detection was proposed. In³⁺ ion was chelated with alizarin red S to form a complex In‐ARS. The complex was used as the template molecule to prepare a molecularly imprinted polymer (MIP) based sensor. The selectivity of the sensor was improved significantly due to the three‐dimensional specific structure of the complex, and the selective complexation of ligands for metal ions. Moreover, the sensitivity of the proposed sensor was improved by recording the reductive current of ligand in complex. This technique was highly sensitive for quantitative analysis of In³⁺ in the concentrations ranged from 1×10^?8 mol/L to 2.5×10^?7 mol/L with a detection limit of 4.7×10^?9 mol/L. The proposed sensor has been successfully used in detecting In³⁺ in real samples.     

Improved conditions for fluorescent staining of proteins with 4,4′‐dianilino‐1,1′‐binaphthyl‐5,5′‐disulfonic acid in SDS‐PAGE

A simple and sensitive fluorescent staining method for the detection of proteins in SDS‐PAGE, namely IB (improved 4,4′‐dianilino‐1,1′‐binaphthyl‐5,5′‐disulfonic acid) stain, is described. Non‐covalent hydrophobic probe 4,4′‐dianilino‐1,1′‐binaphthyl‐5,5′‐disulfonic acid was applied as a fluorescent dye, which can bind to hydrophobic sites in proteins non‐specifically. As low as 1 ng of protein band can be detected briefly by 30 min washing followed by 15 min staining without the aiding of stop or destaining step. The sensitivity of the new presented protocol is similar to that of SYPRO Ruby, which has been widely accepted in proteomic research. Comparative analysis of the MS compatibility of IB stain and SYPRO Ruby stain allowed us to address that IB stain is compatible with the downstream of protein identification by PMF.     

Highly Sensitive Voltammetric Speciation and Determination of Inorganic Arsenic in Water and Alloy Samples Using Ammonium 2‐Amino‐1‐Cyclopentene‐1‐Dithiocarboxylate

A simple, fast, reproducible (2.5% RSD at 3.0 μg/L), and sensitive method is described for quantifying As(III) (0.3 μg/L detection limit, 0.5–440 μg/L dynamic range). Anodic stripping voltammetry (ASV) is performed after accumulating arsenic at a mercury film electrode at ?0.350 V vs. Ag/AgCl (saturated KCl) for 20 s in 0.2 M HCl containing 8 μM ammonium 2‐amino‐1‐cyclopentene‐1‐dithiocarboxylate (AACD), without oxygen removal. This is the first report of using AACD in ASV and in electrochemical quantification of As(III). Total arsenic is determined after sodium‐sulfite‐reduction of As(V) to As(III). Interferences are minimal. Method validation involved water and metal alloy samples.     

Amperometric Immunosensor for the Determination of α‐1‐Fetoprotein Based on Core‐Shell‐Shell Prussian Blue‐BSA‐Nanogold Functionalized Interface

In the present work, a newly functional nanoparticle has been prepared to immobilize the protein for the detection of α‐1‐fetoprotein (AFP). Prussian blue (PB) nanoparticle was initially synthesized under ultrasonic condition, then bovine serum albumin (BSA) was used to coat the PB nanoparticle to improve the stability of the PB nanoparticle as well as functionalize the surface of PB nanoparticle, and then gold colloids were loaded on the BSA‐coated PB nanoparticle to construct a core‐shell‐shell nanostructure via the conjunction of thiolate linkages or alkylamines of the BSA. Finally, a convenient, effective and sensitivity amperometric immunosensor for the detection of α‐1‐fetoprotein (AFP) was constructed by the employment of these functional core‐shell‐shell microspheres. The preparation of the nanoparticle (Au‐BSA‐PB NPs) was characterized by transmission electron microscopy (TEM), and the assembly of the biosensor was characterized with cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The dynamic range of the resulted immunosensor for the detection of AFP is from 0.02 ng/mL to 200.0 ng/mL with a detection limit of 0.006 ng/mL (S/N=3). Moreover, this biosensor displays good selectivity, stability and reproducibility.     

Sensitive phosphoprotein detection in SDS‐PAGE via Anthracene Chrome Red A stain

Protein phosphorylation, one of the most important post‐translational modifications, plays critical roles in many biological processes. Thus, it is necessary to precisely detect, identify and understand the phosphoproteins from protein mixture for the study of cell biology. We introduce a sensitive and specific detection method for phosphoproteins in sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE). Anthracene Chrome Red A (ACRA) combined with the trivalent metal ion (Al³⁺) is converted to fluorescent complex and the fluorescence is sharply increased by a change of pH environment. Phosphoproteins and non‐phosphoproteins can be easily distinguished by the fluorescence quenching due to the structural change of ACRA‐Al³⁺‐phosphoprotein complex, unlike non‐phosphoprotein complex. The method using ACRA is a negative staining based on the fluorescence quenching and has a high sensitivity comparable to Pro‐Q Diamond stain. ACRA stain can detect 1–2 ng of α‐casein and β‐casein, 8–16 ng of ovalbumin (OVA) and κ‐casein within 130 min. Moreover, the ACRA stain showed similar linear dynamic ranges and RSD to Pro‐Q stain. The linear dynamic ranges of ACRA and the values of correlation coefficient were for OVA (8–500 ng, correlation coefficient r = 0.999), α‐casein (4–500 ng, r  = 0.992), β‐casein (4–500 ng, r  = 0.996), and κ‐casein (8–500 ng, 0.998), respectively. On the other hand, the values of the relative standard deviations (RSD) ranged from 2.33 to 3.56% for ACRA. The method is sensitive, specific, simple, rapid and compatible with total protein stain such as SYPRO Ruby stain. Therefore, ACRA stain can be an advanced method for phosphoprotein detection in gels.     

Design and synthesis of a novel fluorescent protein probe for easy and rapid electrophoretic gel staining by using a commonly available UV‐based fluorescent imaging system

A new fluorescent molecular probe, methyl 3‐(3,5‐bis((bis(pyridin‐2‐ylmethyl)amino)‐methyl)‐4‐hydroxyphenyl)‐2‐(5‐(dimethylamino)naphthalene‐1‐sulfonamido) propanoate, dizinc(II) chloride salt (Dansyl‐ 1 ‐Zn(II)), which possesses Zn(II) complexes and a dansyl group, was designed and synthesized to enable the detection of proteins in solution and in high‐throughput electrophoresis by using a UV‐based detection system. Dansyl‐ 1 ‐Zn(II) exhibited weak fluorescence in the absence of proteins and strong green fluorescence at approximately 510 nm in the presence of BSA upon irradiation with light at a wavelength of 345 nm. Compared with conventional protocols for in‐gel SDS‐PAGE protein staining (e.g. silver staining, SYPRO Ruby, and Oriole), the operating times of which range from 90 min to overnight, Dansyl‐ 1 ‐Zn(II) allowed 1‐step protein staining (SDS‐PAGE →Staining →Detection) and shortened the operating time (35 min) with high sensitivity (LOD: 1 ng or less) under 312‐nm or 365‐nm light excitation with orange or red emission filters, respectively. Moreover, Dansyl‐ 1 ‐Zn(II) was successfully applied to protein identification by MS via in‐gel tryptic digestion, Western blotting, and Native‐PAGE. Accordingly, Dansyl‐ 1 ‐Zn(II) may facilitate highly sensitive and high‐throughput protein detection, and it may be widely applicable as a convenient tool in various scientific and medical fields.     

A New Colorimetric Platform for Protein Detection Based on Recognition‐Induced Cascade of Polymeric Nanoparticles Disassembly

Despite great progress, it is still of high interest to explore new homogeneous assays for simple, visual, and selective protein detection. Herein, one new colorimetric sensor has been developed for visual detection of protein by using polymeric micelles as a sensing scaffold and the molecular recognition between protein and the ligand on the surface of the polymeric micelles as the driving force to trigger the readout of the detection signal. The polymeric micelles formed via the self‐assembly of the amphiphilic block polymer biotin‐labeled poly(ethylene glycol)‐block‐poly(3‐acryl aminophenylboronic acid) are endowed with colorful feature by incorporation of alizarin red S (ARS) into the hydrophobic core. Based on the response to streptavidin recognition, these micelles are further disintegrated through the competitive binding of α‐cyclodextrin with boronic acid for disassociation of ARS, which achieves orange–yellow to pink–purple transition in 2 h. This work will open the way to develop one new mix‐and‐measure, visual, and homogeneous assay.     

On‐Line Determination of Arsenic Species in Sea Water by Selective Hydride Generation Coupled with Inductively Coupled Plasma — Mass Spectrometry

A method for direct de termination of total in organic arsenic (III+V), arsenic (III) and dimethylarsinate (DMA) in sea water was developed by combining continuous‐flow selective hydride generation and inductively coupled plasma mass spectrometry (ICP‐MS) is presented. The principle underlying selective hydride generation is based on proper control of the reaction conditions for achieving separation of the respective arsenic species. The effects of pH and composition of reaction media on mutual interference between the arsenic species were investigated in detail. The results indicate that the appropriate media for the selective determination of total in organic arsenic, DMA and As(III) are 6 M HNO₃, acetate buffer at pH = 4.63 and citrate buffer at pH = 6.54, respectively. The concentrations of total inorganic arsenic species, As(III+V), and As(III) were respectively deter mined and that of As(V) was obtained by the difference between them. As to the concentration of DMA, it was obtained after correction from the interference caused by As(III) and As(V). By following the established procedure, the detection lim its (as based on 3‐sigma criterion) for As(III+V), As(III) and DMA were 0.050, 0.009, and 0.002 ng/mL, respectively. There liability of the pro posed method was evaluated in terms of precision and spike addition. The results indicated that the precision of better than 3% and spike recovery of 95 to 105% for all the arsenic species tested in the natural sea water samples can be obtained.     

Determination of trace aluminum by fluorescence quenching method based on catalysis of potassium chlorate oxidizing alizarin red

A new method for the determination of trace aluminum has been proposed. It is based on the fact that alizarin red can emit strong and stable fluorescence at 80 degrees C for 30 min and Al(3+) can effectively catalyze potassium chlorate oxidizing alizarin red to form non-fluorescence complex which cause the fluorescence quenching. The linear dynamic range of this method is 0.040-4.00 ngl(-1) with a detection limit of 5.3 pgl(-1). The regression equation can be expressed as DeltaI(f)=8.731+21.73c(Al(3+)) (ngl(-1)), with the correlation coefficient r=0.9992 (n=6). This sensitive, rapid and accurate method has been applied to the determination of trace aluminum(III) in human hair and tea samples successfully. What is more, the mechanism of catalyzing potassium chlorate oxidizing alizarin red by the fluorescence quenching method is also discussed.     

Thulium(III) Ions Monitoring by a Novel Thulium(III) Microelectrode Based on a S‐N Schiff Base

This study presents for the first time development of a highly selective and sensitive thulium(III) micro‐sensor. Theoretical calculations were conducted on a S‐N Schiff base [thiophene‐2‐carbaldehyde‐(7‐methyl‐1,3‐benzothiazol‐2‐yl) hydrazone] (TCMH) in order to obtain a clue about the tendency of TCMH to Tm(III) and some other metal ions. Then, TCMH was used as a membrane‐active component to prepare a Tm(III)‐selective polymeric membrane microelectrode. In line with the resulting data, the electrode exhibits a Nernstian response toward Tm(III) ions for a very wide concentration range (1.0×10^?11–4.0×10^?6 M) with a detection limit of 1.0×10^?11 (ca. 1.5 ppt) and a very fast response time in the whole concentration range (<5 s). In addition, the results showed that the certain microelectrode could be applied in the pH range of 4.0–11.0 with a usage of more than one month without any considerable potential divergence.     

A Novel Al(III)‐Selective Electrochemical Sensor Based on N,N′‐Bis(salicylidene)‐1,2‐phenylenediamine Complexes

A polyvinylchloride membrane sensor based on N,N′‐bis(salecylidene)‐1,2‐phenylenediamine (salophen) as membrane carrier was prepared and investigated as a Al³⁺‐selective electrode. The sensor exhibits a Nernstian response toward Al(III) over a wide concentration range (8.0×10^?7–3.0×10^?2 M), with a detection limit of 6.0×10^?7 M. The potentiometric response of the sensor is independent of the pH of the test solution in the pH range 3.2–4.5. The electrode possesses advantages of very fast response and high selectivity for Al³⁺ in comparison with alkali, alkaline earth and some heavy metal ions. The sensor was used as an indicator electrode, in the potentiometric titration of aluminum ion and in determination of Al³⁺ contents in drug, water and waste water samples.