A fluorescent derivatization method of proteins for the detection of low‐level impurities by microchip capillary gel electrophoresis

A novel pre‐chip fluorescent derivatization method is presented for protein sizing and quantification by microchip CGE. The derivatization reaction employed a water‐soluble and stable fluorescent dye and was performed under conditions that favored the formation of homogeneous reaction products. The method delivered in terms of protein sizing similar results as microchip CGE with on‐chip staining but showed an extended linear dynamic range for protein quantification encompassing four orders of magnitude. The sensitivity of the method was similar to standard silver‐stained planar gels. The characterization of derivatization reaction products by MS and preparative isoelectric focusing indicated that a constant degree of dye molecule tagging was obtained over a broad range of protein/dye ratios. The method allowed detecting and quantifying an impurity spiked into an antibody preparation down to a level of 0.05%. Advantages of this method compared with CGE approaches with pre‐column derivatization include a shorter analysis time and an increased robustness and ease of use.     

Glyoxylic acid in the reaction of isatoic anhydride with amines: a rapid synthesis of 3-(un)substituted quinazolin-4(3H)-ones leading to rutaecarpine and evodiamine

A dual reactant/catalyst role of glyoxylic acid in the reaction of isatoic anhydride with various amines afforded a novel, robust and rapid synthesis of 3-(un)substituted quinazolin-4(3H)-ones. This metal catalyst-free reaction proceeds via an unusual and unexpected cleavage of C–C bond. A shorter and common route to two alkaloids, that is, rutaecarpine and evodiamine is also accomplished.     

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.     

A highly sensitive “turn‐on” fluorescent sensor for the detection of human serum proteins based on the size exclusion of the polyacrylamide gel

A highly sensitive “turn‐on” fluorescent sensor based on the size exclusion of the polyacrylamide gel was developed for the on‐gels detection of human serum proteins after PAGE. The possible mechanism of this fluorescence sensor was illustrated and validated by utilizing five kinds of colloidal silver nanoparticles with different particle size distribution and six kinds of polyacrylamide gels with different pore size. It was attributed to that silver nanoparticles (<5 nm in diameter) had been selectively absorbed into the gel and formed the small silver nanoclusters, resulting in the red fluorescence. Using this new technique for the detection of human serum proteins after PAGE, a satisfactory sensitivity was achieved and some relatively low‐abundance proteins (e.g. zinc‐alpha‐2‐glycoprotein), which are the significant proteinic markers of certain diseases can be easily detected, but not with traditional methods. Furthermore, it was also successfully applied to distinguish between serums from hepatoma patient and healthy people. As a new protein detection technique, the colloidal silver nanoparticles based “turn‐on” fluorescent sensor offers a rapid, economic, low background, and sensitive way for direct detection of human serum proteins, showing available potential and significance in the development of nanobiotechnology and proteome research.     

Postcolumn derivatization of proteins in capillary sieving electrophoresis/laser‐induced fluorescence detection

The separation methods for proteins with high resolution and sensitivity are absolutely important in the field of biological sciences. Capillary sieving electrophoresis (CSE) is an excellent separation technique for DNA and proteins with high resolution, while LIF permits the most sensitive detection in CSE. Therefore, proteins have to be labeled with fluorescent or fluorogenic reagent to produce fluorescent derivatives. Both precolumn and oncolumn derivatization have been employed for the labeling of proteins in CSE. However, there is no report on the postcolumn derivatization due to the limitation in the use of a standard migration buffer, despite it being a promising method for sensitive detection of proteins. Here, we show a novel postcolumn derivatization method for protein separation by CSE, using a tertiary amine as a buffer component in the running buffer. Tris, which is commonly used as a base in CSE separation buffers, was substituted by tertiary amines, 2‐(diethylamino)ethanol and triethanolamine. A buffer solution containing 2‐(diethylamino)ethanol or triethanolamine can be used for the CSE separation followed by the postcolumn derivatization of proteins, since both reagents are unreactive toward a fluorogenic labeling reagent, naphthalene‐2,3‐dicarbaldehyde. Thus, LIF detection using the postcolumn derivatization permits significant reduction in the LOD (by a factor of 2.4–28) of proteins, compared with conventional absorbance detection.     

Design and synthesis of new fluorescent probe for rapid and highly sensitive detection of proteins via electrophoretic gel stain

A new fluorescent molecular probe, 2,2′‐(1E,1′E)‐2,2′‐(4‐(dicyanomethylene)‐4H‐pyrane‐2,6‐diyl)bis(ethene‐2,1‐diyl)bis(sodium benzenesulfonate) salt ( 1 ), possessing the cyanopyranyl moieties and two benzene sulfonic acid groups was designed and synthesized to detect proteins in solution and for high‐throughput SDS‐PAGE. Compound 1 exhibited no fluorescence in the absence of proteins; however, it exhibited strong fluorescence on the addition of bovine serum albumin as a result of intramolecular charge transfer. Compared with the conventional protocols for in‐gel protein staining, such as SYPRO Ruby and silver staining, 1 achieves higher sensitivity, even though it offers a simplified, higher throughput protocol. In fact, the total time required for protein staining was 60–90 min under optimum conditions much shorter than that required by the less‐sensitive silver staining or SYPRO Ruby staining protocols. Moreover, 1 was successfully applied to protein identification by mass spectrometry via in‐gel tryptic digestion, Western blotting, and native PAGE together with protein staining by 1 , which is a modified protocol of blue native PAGE (BN‐PAGE). Thus, 1 may facilitate high‐sensitivity protein detection, and it may be widely applicable as a convenient tool in various scientific and medical fields.     

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.     

Determination of multiple phytohormones in fruits by high‐performance liquid chromatography with fluorescence detection using dispersive liquid–liquid microextraction followed by precolumn fluorescent labeling

Plant hormone determination in food matrices has attracted more and more attention because of their potential risks to human health. However, analytical methods for the analysis of multiple plant hormones remain poorly investigated. In the present study, a convenient, selective, and ultrasensitive high‐performance liquid chromatography method for the simultaneous determination of multiple classes of plant hormones has been developed successfully using dispersive liquid–liquid microextraction followed by precolumn fluorescent labeling. Eight plant hormones in fruits including jasmonic acid, 12‐oxo‐phytodienoic acid, indole‐3‐acetic acid, 3‐indolybutyric acid, 3‐indolepropionic acid, gibberellin A₃, 1‐naphthylacetic acid, and 2‐naphthaleneacetic acid were analyzed by this method. The conditions employed for dispersive liquid–liquid microextraction were optimized systematically. The linearity for all plant hormones was found to be >0.9993 (R² values). This method offered low detection limits of 0.19–0.44 ng/mL (at a signal‐to‐noise ratio of 3), and method accuracies were in the range of 92.32–103.10%. The proposed method was applied to determine plant hormones in five kinds of food samples, and this method can achieve a short analysis time, low threshold levels of detection, and a high specificity for the analysis of targeted plant hormones present at trace level concentrations in complex matrices.     

Rapid labeling of amino acid neurotransmitters with a fluorescent thiol in the presence of o‐phthalaldehyde

LIF detection often requires labeling of analytes with fluorophores; and fast fluorescent derivatization is valuable for high‐throughput analysis with flow‐gated CE. Here, we report a fast fluorescein‐labeling scheme for amino acid neurotransmitters, which were then rapidly separated and detected in flow‐gated CE. This scheme was based on the reaction between primary amines and o‐phthalaldehyde in the presence of a fluorescent thiol, 2‐((5‐fluoresceinyl)aminocarbonyl)ethyl mercaptan (FACE‐SH). The short reaction time (<30 s) was suited for on‐line mixing and derivatization that was directly coupled with flow‐gated CE for rapid electrophoretic separation and sensitive LIF detection. To maintain the effective concentration of reactive FACE‐SH, Tris(2‐carboxyethyl)phosphine was added to the derivatization reagents to prevent thiol loss due to oxidation. This labeling scheme was applied to the detection of neurotransmitters by coupling in vitro microdialysis with online derivatization and flow‐gated CE. It is also anticipated that this fluorophore tagging scheme would be valuable for on‐chip labeling of proteins retained on support in SPE.     

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 new evaluation technique for the detection of impurities in purified proteins via CE with native UV‐LIF

An analytical methodology for quality control analyses of IgG and their impurities is presented using a new UV‐LIF (266 nm) detector inside the cassette of a CE instrument and its performance was evaluated. The observed sensitivity was very close to that obtained by silver staining of slab gels (LOD of 25 ng/mL), while the sensitivity of the analysis is 80 times better than with CE/UV absorption (214 nm). Examples of the analysis of pharmaceutical and other commercial IgGs are provided and the kinetics of the reduction of IgG by β‐mercaptoethanol is reported, demonstrating the ease of performing the analysis.     

Improved dynamic range of protein quantification in silver‐stained gels by modelling gel images over time

Silver staining is a commonly used protein stain to visualise proteins separated by 2‐DE. Despite this, the technique suffers from a limited dynamic range, making the simultaneous quantification of high‐ and low‐abundant proteins difficult. In this paper we take advantage of the fact that silver staining is not an end‐point stain by photographing the gels during development. This procedure provides information about the change in measured absorbance for each pixel in the protein spots on the gel. The maximum rate of change was found to be correlated with the amount of applied protein, providing a new way of estimating protein amount in 2‐DE gels. We observed an improvement in the dynamic range of silver staining by up to two orders of magnitude.     

Fabrication and characterization of implantable flushable electrodes for electric field‐mediated drug delivery in a brain tissue‐mimic agarose gel

Every forty minutes, one person dies in the USA due to glioblastoma multiforme; a deadly form of brain cancer with an average five‐year survival rate less than 3%. The current standard of care for treatment involves surgical resection of the accessible tumor followed by radiation therapy and concomitant chemotherapy. Despite their potency, delivering chemotherapeutic agents to the brain is limited by the highly selective blood‐brain barrier, which prevents molecules >500 Da from reaching the brain. Other techniques, such as convection‐enhanced delivery, controlled release by drug‐loaded wafers or intracerebroventricular infusion have limited clinical utility due to unpredictable targeting and volume of drug distribution. We introduce a novel drug delivery technique that can use direct current electric fields to deliver charged chemotherapeutics to the site of brain parenchyma after tumor resection. We fabricate and characterize an implantable drug delivery system using flushable electrodes to deliver the charged chemotherapeutic or doxorubicin (+1) in a brain tissue‐mimic agarose gel (0.2% w/v) model by electrophoresis. The optimized capillary‐embedded electrode system exhibited a sustained movement of charged doxorubicin through nearly 3.5 mm in four hours, a distance for achieving effective intratumoral concentrations.     

Discriminative detection of low‐abundance point mutations using a PCR/ligase detection reaction/capillary gel electrophoresis method and fluorescence dual‐channel monitoring

We applied a facile LIF dual‐channel monitoring system recently developed and reported by our group to the polymerase chain reaction/ligase detection reaction/CGE method for detecting low‐abundance point mutations present in a wild‐type sequence‐dominated population. Mutation discrimination limits and signaling fidelity of the analytical system were evaluated using three mutant variations in codon 12 of the K‐ras oncogene that have high diagnostic value for colorectal cancer. We demonstrated the high sensitivity of the present method by detecting rare mutations present among an excess of wild‐type alleles (one mutation among ～100 normal sequences). This method also simultaneously interrogated the allelic compositions of the test samples with high specificity through spectral discrimination of the dye‐tagged ligase detection reaction products using the dual‐channel monitoring system.     

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.     

Preparation of poly(methyl acrylate‐co‐itaconic anhydride)/SiO2 hybrid materials via the sol–gel process—The effect of the coupling agent,inorganic content,and nature of the catalyst

Transparent poly(methyl acrylate‐co‐itaconic anhydride)/SiO₂ hybrid materials were prepared from methyl acrylate‐itaconic anhydride copolymer and tetraethoxysilane (TEOS) with the coupling agent (3‐aminopropyl)triethoxysilane (APTES) via a sol–gel process. The covalent bonds between the organic and inorganic phases were introduced by the in situ aminolysis of the itaconic anhydride units with APTES forming a copolymer bearing a triethoxysilyl group. These groups subsequently were hydrolyzed with TEOS and allowed to form a network. These reactions were monitored by Fourier transform infrared analysis. The amount of APTES had a dramatic influence on the gel time and sol fraction. The effect of APTES, the inorganic content, and the nature of the catalyst on the thermal properties and morphology of the hybrid materials were studied by differential scanning calorimetry, thermogravimetric analysis, scanning electron microscopy, and atomic force microscopy. © 2000 John Wiley & Sons, Inc. J Polym Sci A: Polym Chem 38: 321–328, 2000     

Optimized sample preparation strategy for the analysis of low molecular mass adducts of a fluorescent cisplatin analogue in cancer cell lines by CE‐dual‐LIF

Pt‐based anticancer drugs, such as cisplatin, are known to undergo several (bio‐)chemical transformation steps after administration. Hydrolysis and adduct formation with small nucleophiles and larger proteins are their most relevant reactions on the way to the final reaction site (DNA), but there are still many open questions regarding the identity and pharmacological relevance of various proposed adducts and intermediates. Furthermore, the role of buffer components or additives, which are inevitably added to samples during any type of analytical measurement, has been frequently neglected in previous studies. Here, we report on adduct formation reactions of the fluorescent cisplatin analogue carboxyfluorescein diacetate platinum (CFDA‐Pt) in commonly used buffers and cell culture medium. Our results indicate that chelation reactions with noninnocent buffers (e.g., Tris) and components of the cell culture/cell lysis medium must be taken into account when interpreting results. Adduct formation kinetics was followed up to 60 h at nanomolar concentrations of CFDA‐Pt by using CE‐LIF. CE‐MS enabled the online identification of such unexpected adducts down to the nanomolar concentration range. By using an optimized sample preparation strategy, unwanted adducts can be avoided and several fluorescent adducts of CFDA‐Pt are detectable in sensitive and cisplatin‐resistant cancer cell lines. By processing samples rapidly after incubation, we could even identify the initial, but transient, Pt species in the cells as deacetylated CFDA‐Pt with unaltered complexing environment at Pt. Overall, the proposed procedure enables a very sensitive and accurate analysis of low molecular mass Pt species in cancer cells, involving a fast CE‐LIF detection within 5 min.     

Fluorescence energy transfer-sensitized photobleaching of a fluorescent label as a tool to study donor-acceptor distance distributions and dynamics in protein assemblies: studies of a complex of biotinylated IgM with streptavidin and aggregates of concanavalin A

A photokinetic method of detection of fluorescence resonance energy transfer (FRET) between special fluorescent labels is applied to study time-averaged spatial distribution of labeled proteins in protein assemblies. Prolonged irradiation of a sample at the absorption maximum of the energy donor initiates FRET-sensitized fluorescence photobleaching of the energy acceptor label, which was monitored by steady-state fluorimetric measurements. Kinetics of the acceptor photobleaching and kinetics of decreasing the efficiency of FRET from donors to unbleached acceptors were determined. The FRET efficiency was found from measuring sensitization of acceptor fluorescence. Analysis of the photokinetic data permits to estimate the time-averaged distribution of acceptors on donor-acceptor distances in the range of characteristic distances of FRET. Dynamic processes influencing donor-acceptor distances can be also investigated by the method. Application of the method is demonstrated by the studies of a complex of biotinylated IgM with streptavidin and aggregates composed of concanavalin A and sodium dodecyl sulphate. A new thiadicarbocyanine dye was used as the acceptor label. R-phycoerythrin and tetramethylrhodamine isothiocyanate were the donor labels. In the IgM-streptavidin complex, 16% of acceptors most contributed to FRET provided 90% of FRET efficiency, whereas acceptors made about the same time-averaged contribution to FRET in the concanavalin A aggregates.