Pui-Kin So

A Highly Selective Luminescent Switch‐On Probe for Histidine/Histidine‐Rich Proteins and Its Application in Protein Staining

The luminescence sensing of histidine and histidine-rich proteins plays a pivotal role in biochemistry and molecular biology, in particular when both temporal and spatial resolution are required. An abnormal level of histidine-rich proteins is an indicator for many diseases, such as advanced liver cirrhosis, AIDS, renal disease, asthma, pulmonary disorders, thrombotic disorders, f] and malaria. Some analyses of histidine and histidine-rich proteins have been developed in conjunction with immunoassay and colorimetric detection methods. The most commonly used method for the detection of histidine and histidine-rich proteins in biological samples is chromatography, which is usually performed through the combination of an effective separation technique, such as thin-layer chromatography, gas chromatography, or HPLC, followed by UV/Vis or fluorescence spectroscopy. The use of high-performance capillary electrophoresis with a derivation reagent has also been reported. However, the aforementioned methods are generally tedious, laborious, and, most importantly, expensive for routine detection in a biochemistry laboratory. Although numerous studies have dealt with the detection of histidine or histidine-rich proteins, studies on the use of luminescent probes for this purpose remain sparse. Notable examples include research by Fabbrizzi and co-workers, who developed competitive noncovalent fluorescence turn-on probes for histidine in the form of dizinc(II) or dicopper(II) macrocyclic complexes, which recognize histidine through the formation of an imidazolate bridge between the two dizinc(II) or dicopper(II) centers; however, the resulting noncovalent ensemble may be less stable than a covalently linking sensory system, and the complexity of the synthetic process makes it difficult to implement in a convenient manner. Photoluminescent iridium(III) complexes have emerged as a topical area of interest in inorganic photochemistry and phosphorescent materials for optoelectronic and luminescence signaling applications. Significant changes in the photophysical behavior and emission properties of iridium(III) complexes may be induced by the presence of biomolecules. Luminescent transition-metal complexes for protein staining, such as the luminescent ruthenium complex known as SYPRO Ruby dye, have been reported previously. However, despite its high sensitivity and its broad dynamic range, the use of SYPRORuby dye is limited, as it is sold only as a formulated solution; therefore, it is not possible to optimize the dye for a particular electrophoresis protocol and protein. In this context, the luminescent cyclometalated iridium(III) solvent complex [Ir(ppy)2(solv)2] + (1; ppy= 2phenylpyridine, solv=H2O or CH3CN) has received particular attention for the following reasons: 1) [Ir(ppy)2(OH2)2] , which contains weakly bound solvent ligands, may bind covalently to amino acids/proteins through a ligand substitution reaction with the OH2 ligand; 2) an intriguing solvent/ media dependence of the emission properties of [Ir(ppy)2(OH2)2] + has been observed; 3) [Ir(ppy)2(OH2)2] + can be synthesized conveniently and rapidly; 4) the use of organic solvents is not required for the optimal sensing of amino acids/proteins with [Ir(ppy)2(OH2)2] , and the iridium complex is readily soluble and stable in aqueous staining solutions. In this study, [Ir(ppy)3] (2) was also prepared for comparative studies, as its binding with proteins was expected to be largely hydrophobic in nature. Herein, we describe the luminescent switch-on probe [Ir(ppy)2(solv)2] + (1) for histidine/histidinerich proteins and demonstrate its utility in protein staining. The Ir complexes 1-CF3SO3 and 2 (Figure 1a) were prepared according to a previously reported method. The structure of 1-ClO4 was established by X-ray crystallography and is depicted in Figure 1b, and the crystal-packing diagrams are given in the Supporting Information. The metal–ligand bonding parameters for 1-ClO4 are comparable to those reported previously for cyclometalated iridium(III) complexes. The complex 1-CF3SO3 (50 mm) is weakly emissive in phosphate buffered saline (PBS). In the presence of histidine (His), 1-CF3SO3 exhibits an intense emission at lmax= 505 nm, the intensity of which reaches saturation level at [His]/[Ir] 4 (Figure 2a). A plot of I/I0 versus [His]/[1-CF3SO3] (I and I0 are emission intensities with and without His) shows an up-to180-fold intensity enhancement at ratios [His]/[1-CF3SO3] 4:1. The luminescence response of 1-CF3SO3 to various other [*] Dr. D.-L. Ma, Dr. W.-L. Wong, W.-H. Chung, F.-Y. Chan, P.-K. So, Dr. T.-S. Lai, Z.-Y. Zhou, Prof. Y.-C. Leung, Prof. K.-Y. Wong Department of Applied Biology and Chemical Technology, Central Laboratory of the Institute of Molecular Technology for Drug Discovery and Synthesis, The Hong Kong Polytechnic University Hung Hom, Kowloon, Hong Kong (China) Fax: (+1)852-2364-9932 E-mail: bcedmond@polyu.edu.hk bckywong@inet.polyu.edu.hk

Electrospray Ionization Using Wooden Tips

Electrospray ionization (ESI) is a mass spectrometric technique widely used in various fields including chemistry, biology, medicine, pharmaceutical industry, clinical assessment, and forensic science. In this study, we report a simple and economical ESI-mass spectrometry (MS) technique, which makes use of disposable wooden tips (wooden toothpicks) for loading and ionization of samples. Samples could be loaded by normal pipetting onto the tip or simply dipping the tip into sample solutions. The hydrophilic and porous nature of wood allows effective adhesion of the sample solution for durable ion signals. The tip can be directly connected to nano-ESI ion sources of various mass spectrometers. Upon application of high voltage to the tip, desirable mass spectra could be obtained. We demostrated that this new technique is applicable for analysis of various samples, including organic compounds, organometallic compounds, peptides, proteins, and samples that cannot be directly analyzed by conventional ESI techniques, e.g., slurry samples and powder samples. The slim and hard properties of the wooden tip enable sampling from specific locations such as corners and small openings, indicating potential applications of the new technique in forensic investigations. The observation of electrospray ionization from wooden materials also allows us to get new insights into the materials that can be directly ionized for mass spectrometric analysis.

Pegylated derivatives of recombinant human arginase (rhArg1) for sustained in vivo activity in cancer therapy: preparation, characterization and analysis of their pharmacodynamics in vivo and in vitro and action upon hepatocellular carcinoma cell (HCC)

BackgroundProtein used in medicine, e.g. interferon, are immunogenic and quickly broken down by the body. Pegylation is a recognized way of preserving their integrity and reducing immune reactions, and works well with enzymes used to degrade amino acids, a recent focus of attention in controlling cancer growth. Of the two arginine-degrading enzymes being explored clinically, arginine deiminase is a decidedly foreign mycoplasm-derived enzyme, whereas human arginase 1 is a native liver enzyme. Both have been pegylated, the former with adjuncts of 20 kD, the latter with 5 kD PEG. Pegylation is done by several different methods, not all of which are satisfactory or desirable.MethodsThe preparation of novel polyethylene glycol (PEG) derivatives for modifying proteins is described, but directed specifically at pegylation of recombinant human arginase 1 (rhArg1). rhArg1 expressed in Escherichia coli was purified and coupled in various ways with 5 different PEG molecules to compare their protective properties and the residual enzyme activity, using hepatocellular cell lines both in vitro and in vivo.ResultsMethoxypolyethylene glycol-succinimidyl propionate (mPEG-SPA 5,000) coupled with very high affinity under mild conditions. The resulting pegylated enzyme (rhArg1-peg5,000 mw) had up to 6 PEG chains of 5K length which not only protected it from degradation and any residual immunogenicity, but most importantly let it retain >90% of its native catalytic activity. It remained efficacious in depleting arginine in rats after a single ip injection of 1,500 U of the conjugate as the native enzyme, plasma arginine falling to >0.05 μM from ~170 μM within 20 min and lasting 6 days. The conjugate had almost the same efficacy as unpegylated rhArg1 on 2 cultured human liver cancer (HCC) cell lines. It was considerably more effective than 4 other pegylated conjugates prepared.ConclusionValuable data on the optimization of the pegylation procedure and choice of ligand that best stabilizes the enzyme arginase 1 are presented, a protocol that should equally fit many other enzymes and proteins. It is a long lasting arginine-depleting enzyme in vivo which will greatly improve its use in anti-cancer therapy.

Analytical properties of solid-substrate electrospray ionization mass spectrometry.

AbstractConventional electrospray ionization mass spectrometry (ESI-MS) uses a capillary for sample loading and ionization. Along with the development of ambient ionization techniques, ESI-MS using noncapillary emitters has attracted more interest in recent years. Following our recent report on ESI-MS using wooden tips (Anal. Chem. 83, 8201–8207 (2011)), the technique was further investigated and extended in this study. Our results revealed that the wooden tips could serve as a chromatographic column for separation of sample components. Sequential and exhaustive ionization was observed for proteins and salts on wooden tips with salts ionized sooner and proteins later. Nonconductive materials that contain microchannels/pores could be used as tips for ESI-MS analysis with sample solutions loaded to the sharp-ends only, since rapid diffusion of sample solutions by capillary action would enable the tips to become conductive. Tips of inert materials such as bamboo, fabrics, and sponge could be used for sample loading and ionization, while samples such as tissue, mushroom, and bone could form tips to induce ionization for direct analysis with application of a high voltage. Figure

Rapid detection and quantitation of ketamine and norketamine in urine and oral fluid by wooden-tip electrospray ionization mass spectrometry

Drug analysis is an indispensable task in controlling drug abuse, which is a serious problem worldwide nowadays. In this study, we report a simple and rapid approach for detection and quantitation of drugs-of-abuse in urine and oral fluid by wooden-tip electrospray ionization mass spectrometry (WT-ESI-MS). We demonstrated that ketamine, one of the most common abused drugs, and its major metabolite, norketamine, in raw urine and oral fluid could be readily detected and quantified by WT-ESI-MS with only little sample preparation and no chromatographic separation, and the analytical performances, including the linear range, accuracy, precision, LOD and LOQ, were well acceptable for analysis of real samples.

Electrospray ionization with aluminum foil: A versatile mass spectrometric technique

In this study, we developed a novel electrospray ionization (ESI) technique based on household aluminum foil (Al foil) and demonstated the desirable features and applications of this technique. Al foil can be readily cut and folded into desired configuration for effective ionization and for holding sample solution in bulk to allowing acquisition of durable ion signals. The present technique was demonstrated to be applicable in analysis of a wide variety of samples, ranging from pure chemical and biological compounds, e.g., organic compounds and proteins, to complex samples in liquid, semi-solid, and solid states, e.g., beverages, skincare cream, and herbal medicines. The inert, hydrophobic and impermeable surface of Al foil allows convenient and effective on-target extraction of solid samples and on-target sample clean-up, i.e., removal of salts and detergents from proteins and peptides, extending ESI device from usually only for sample loading and ionization to including sample processing. Moreover, Al foil is an excellent heat-conductor and highly heat-tolerant, permitting direct monitoring of thermal reactions, e.g., thermal denaturation of proteins. Overall, the present study showed that Al-foil ESI could be an economical and versatile method that allows a wide range of applications.

A general strategy for site-directed enzyme immobilization by using NiO nanoparticle decorated mesoporous silica.

Mesoporous materials have recently gained much attention owing to their large surface area, narrow pore size distribution, and superior pore structure. These materials have been demonstrated as excellent solid supports for immobilization of a variety of proteins and enzymes for their potential applications as biocatalysts in the chemical and pharmaceutical industries. However, the lack of efficient and reproducible methods for immobilization has limited the activity and recyclability of these biocatalysts. Furthermore, the biocatalysts are usually not robust owing to their rapid denaturation in bulk solvents. To solve these problems, we designed a novel hybrid material system, mesoporous silica immobilized with NiO nanoparticles (SBA-NiO), wherein enzyme immobilization is directed to specific sites on the pore surface of the material. This yielded the biocatalytic species with higher activity than free enzyme in solution. These biocatalytic species are recyclable with minimal loss of activity after several cycles, demonstrating an advantage over free enzymes.

Direct analysis of herbal powders by pipette-tip electrospray ionization mass spectrometry

Conventional electrospray ionization mass spectrometry (ESI-MS) is widely used for analysis of solution samples. The development of solid-substrate ESI-MS allows direct ionization analysis of bulky solid samples. In this study, we developed pipette-tip ESI-MS, a technique that combines pipette tips with syringe and syringe pump, for direct analysis of herbal powders, another common form of samples. We demonstrated that various herbal powder samples, including herbal medicines and food samples, could be readily online extracted and analyzed using this technique. Various powder samples, such as Rhizoma coptidis, lotus plumule, great burdock achene, black pepper, Panax ginseng, roasted coffee beans, Fructus Schisandrae Chinensis and Fructus Schisandrae Sphenantherae, were analyzed using pipette-tip ESI-MS and quality mass spectra with stable and durable signals could be obtained. Both positive and negative ion modes were attempted and various compounds including amino acids, oligosaccharides, glycosides, alkaloids, organic acids, ginosensides, flavonoids and lignans could be detected. Principal component analysis (PCA) based on the acquired mass spectra allowed rapid differentiation of closely related herbal species.

Mass spectrometry: towards in vivo analysis of biological systems

In vivo analysis is of paramount importance in monitoring physiological processes that take place in living organisms. Mass spectrometry, an analytical technique with high speed, sensitivity and specificity, is indispensable in biochemical studies nowadays. However, traditional mass spectrometric techniques are of limited applicability in direct analysis of living organisms due to various constraints, e.g., the necessity of ionization of analytes under vacuum and perturbation of physiological functions of living organisms during analysis. Recent development of mass spectrometry, particularly the development of ambient ionization techniques, has opened the door for direct analysis of living organisms. These new mass spectrometric techniques have the features that the ionization processes take place under atmospheric pressure and no or only little sample preparation is required, thus are well suited for analysis of living specimens without significantly perturbing their physiological states. The role of these mass spectrometric techniques in in vivo analysis has been increasingly important in recent years and is expected to be further expanded in the future. In this review, the use of various mass spectrometric techniques in in vivo analysis of biological systems is summarized and the prospects are discussed.

Rapid differentiation of Panax ginseng and Panax quinquefolius by matrix-assisted laser desorption/ionization mass spectrometry.

A matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS)-based method has been developed for rapid differentiation between Panax ginseng and Panax quinquefolius, two herbal medicines with similar chemical and physical properties but different therapeutic effects. This method required only a small quantity of samples, and the herbal medicines were analyzed by MALDI-MS either after a brief extraction step, or directly on the powder form or small pieces of raw samples. The acquired MALDI-MS spectra showed different patterns of ginsenosides and small chemical molecules between P. ginseng and P. quinquefolius, thus allowing unambiguous differentiation between the two Panax species based on the specific ions, intensity ratios of characteristic ions or principal component analysis. The approach could also be used to differentiate red ginseng or P. quinquefolius adulterated with P. ginseng from pure P. ginseng and pure Panax quinquefolium. The intensity ratios of characteristic ions in the MALDI-MS spectra showed high reproducibility and enabled quantitative determination of ginsenosides in the herbal samples and percentage of P. quinquefolius in the adulterated binary mixture. The method is simple, rapid, robust, and can be extended for analysis of other herbal medicines.