Capillary isoelectric focusing with UV-induced fluorescence detection

Low-molecular-mass fluorescent compounds excitable in the near UV region with suitable acidobasic and electrophoretic properties are suggested as isoelectric point (pI) markers for isoelectric focusing (IEF) with UV photometric and UV excited fluorometric detection. The experimental set-up of capillary IEF with UV excited fluorometric detection and properties of new UV-induced fluorescent pI markers are given. The pI values of 18 new pI markers determined independently of IEF methods range from 2.1 to 10.3. The examples of separation of new pI markers together with derivatized proteins by capillary IEF with photometric or fluorometric detection are presented.     

Fluorescein-based pI markers for capillary isoelectric focusing with laser-induced fluorescence detection

We prepared a series of low-molecular-mass fluorescent ampholytes with narrow pI range. These fluorescein-based ampholytes are detection compatible with argon laser-induced fluorescence (LIF) detection. The selected properties, important for their routine use as fluorescent pI markers, were examined. The pI values of new fluorescein-based pI markers were determined by capillary isoelectric focusing (CIEF) using currently available low-molecular-mass pI markers for CIEF with photometric detection. The examples of CIEF with fluorometric detection of new compounds together with fluorescein isothiocyanate (FITC) derivatized proteins are presented.     

Acousto-optical deflection-based whole channel scanning for microchip isoelectric focusing with laser-induced fluorescence detection

This paper describes the development of a technique amenable to the separation of proteins on a microchip by isoelectric focusing (IEF) with entire channel scanning laser-induced fluorescence detection using acousto-optical deflection (AOD). The ability to use AOD to scan the portions of or the entire length of an IEF separation channel allows for high-speed analysis since the mobilization step is circumvented with this technique. Employing no moving parts eliminates mechanical noise and, not only is there no loss of resolution, AOD scanning can potentially increase resolution. The ability of AOD to provide ultra-fast scanning rates (kHz timescale) allows for real-time imaging of the focusing process. This is demonstrated with the separation of naturally fluorescent proteins using entire channel (total scanning range of 2.4 cm) AOD-mediated scanning laser-induced fluorescence detection.     

Use of a native affinity ligand for the detection of G proteins by capillary isoelectric focusing with laser-induced fluorescence detection

Affinity probe capillary isoelectric focusing (CIEF) with laser-induced fluorescence was explored for detection of Ras-like G proteins. In the assay, a fluorescent BODIPY FL GTP analogue (BGTPgammaS) and G protein were incubated resulting in formation of BGTPgammaS-G protein complex. Excess BGTPgammaS was separated from BGTPgammaS-G protein complex by CIEF using a 3-10 pH gradient and detected in whole-column imaging mode. In other cases, a single point detector was used to detect zones during the focusing step of CIEF using a 2.5-5 pH gradient. In this case, analyte peaks passed the detector in approximately 5 min at an electric field of 350 V/cm. Detection during focusing allowed for more reproducible assays at shorter times but with a sacrifice in sensitivity compared to detection during mobilization. Resolution was adequate to separate BGTPgammaS-Ras and BGTPgammaS-Rab3A complexes. Formation of specific complexes was confirmed by adding GTPgammaS to samples containing BGTPgammaS-G protein. GTPgammaS competed with BGTPgammaS for G protein binding sites resulting in decreased BGTPgammaS-G protein peak heights. The concentrating effect of CIEF enabled detection limits of 30 pM.     

Capillary electrophoresis of aminoglycosides with argon-ion laser-induced fluorescence detection

A new analytical method for aminoglycosides (kanamycin, bekanamycin, paromomycin and tobramycin) based on capillary electrophoretic separation and argon-ion laser-induced fluorescence detection has been developed. 6-Carboxyfluorescein succinimidyl ester (CFSE) was used for precolumn derivatization of the non-fluorescent aminoglycosides. Optimal separation and detection were obtained with an electrophoretic buffer of 30 mM sodium borate (pH 9.0) and an air-cooled argon-ion laser (excitation at 488 nm, emission at 520 nm). The concentration limits of detection in aqueous solution were 3.9-8.2 nM. Combined with a simple cleanup procedure, this method can be applied to the determination of aminoglycosides in human plasma. A calibration curve ranging from 0.15 to 30 microM was shown to be linear. The limits of detection of aminoglycosides in human plasma were between 14.4 and 24.0 nM. Recoveries of spiked aminoglycosides in human plasma were between 92 and 105%.     

Comprehensive two-dimensional separation system by coupling capillary reverse-phase liquid chromatography to capillary isoelectric focusing for peptide and protein mapping with laser-induced fluorescence detection

A comprehensive two-dimensional (2-D) separation system, coupling capillary reverse-phase liquid chromatography (cRPLC) to capillary isoelectric focusing (CIEF), is described for protein and peptide mapping. cRPLC, the first dimension, provided high-resolution separations for salt-free proteins. CIEF, the second dimension with an orthogonal mechanism to cRPLC afforded excellent resolution capability for proteins with efficient protein enrichment. Since all sample fractions in cRPLC effluents could be transferred to the CIEF dimensions, the combination of the two high-efficiency separations resulted in maximal separation capabilities of each dimension. Separation effectiveness of this approach was demonstrated using complex protein/peptide samples, such as yeast cytosol and a BSA tryptic digest. A peak capacity of more than 10 000 had been achieved. A laser-induced fluorescence (LIF) detector, developed for this system, allowed for high-sensitive detection, with a fmol level of peptide detection for the BSA digest. FITC and BODIPY maleimide were used to tag the proteins, and the latter was found better both for separation and detection in our 2-D system.     

A highly sensitive method for quantification of myosin light chain phosphorylation by capillary isoelectric focusing with laser-induced fluorescence detection

Activation of myosin II by phosphorylation of the 20 kDa regulatory light chains (LC20) has been implicated in numerous contractile and motile events, e.g., smooth muscle contraction, cytokinesis, and cell migration. The ability to analyze LC20 phosphorylation in minute samples is critical to determine the importance of LC20 phosphorylation in diverse physiological processes. We have developed a method for the separation and quantification of unphosphorylated, monophosphorylated, and diphosphorylated LC20 with a detection limit of 1 pg (50 amol). LC20 is initially isolated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transblotted to a polyvinlyidene difluoride (PVDF) membrane. The region of the membrane containing the LC20 band (identified by electrophoresis of purified LC20 in a neighboring lane) is cut out and fluorescently labeled with Alexa Fluor 488 C5 maleimide. The labeled LC20 is eluted from the membrane with detergent and subjected to capillary isoelectric focusing (CIEF) to separate unphosphorylated, mono-, and diphosphorylated LC20, which are detected and quantified by laser-induced fluorescence (LIF). A linear relationship between log(peak area) and log(LC20 amount) is observed over the range of 50 amol-150 fmol. Quantification of LC20 phosphorylation by CIEF with LIF detection was compared with three commonly used methods with much lower levels of sensitivity: urea/glycerol-PAGE with Western blotting, phosphorylation by [gamma-32P]ATP with Cerenkov counting, and phosphorylation by [gamma-32P]ATP followed by SDS-PAGE, autoradiography, and scanning densitometry. All four methods gave very similar quantitative results, the major difference being that the new method exhibits 3000-fold enhanced sensitivity. This method is therefore applicable to quantitative analysis of phosphorylation of minute quantities of LC20.     

Capillary electrophoretic competitive immunoassay with laser-induced fluorescence detection for methionine-enkephalin

Immunoassays are commonly used in bioresearch for the detection and quantification of small proteins and macromolecules in biological fluids and other complex matrices. In this report, a competitive immunoassay using capillary electrophoresis (CE) with laser-induced fluorescence was developed for methionine-enkephalin (ME). The method is based on the competitive reaction between the ME and fluorescein conjugated ME (ME-F) with anti-ME antibody, capillary electrophoresis separation of the ME-antibody bound and free ME-F, followed by the laser-induced fluorescence detection of the fluorescent species. With the optimized separation conditions, it was possible to separate the antibody bound and free fluorescien conjugated ME by a capillary electrophoresis-laser-induced fluorescence (CE-LIF) analysis using an uncoated fused-silica capillaries. The results concluded that the assay specificity, selectivity and accuracy were excellent.     

Microchip isoelectric focusing using a miniature scanning detection system

A miniature scanning fluorescent detector has been developed for plastic microchannel isoelectric focusing (mIEF) analysis. The detector, comprised of a lamp and photomultiplier tube (PMT) on a moving stage, measured the real-time distribution of fluorescently labeled peptides subjected to gel-free mIEF. During the run, the effective length of the 6-cm channel was scanned every 9 s. Analysis was completed within 5 min while still obtaining high resolution and sensitivity. In addition, the scanning detector was used to characterize peptide migration properties within the channel by providing simultaneous temporal and spatial measurements.     

Capillary electrophoresis of FITC labeled amino acids with laser-induced fluorescence detection

FITC labeled amino acids have been separated using a home-huilt capillary electrophoresis with a laserinduced fluorescence detection (CE-LIF) system. Seventeen peaks can now be generated from the twenty common amino acids. The key conditions lie in the optimization of pH, buffer electrolytes and buffer additives.     

Capillary electrophoresis with laser-induced fluorescence detection for ATP quantification in spermatozoa and oocytes

We describe a new capillary electrophoresis laser-induced fluorescence (CE-LIF) method for the quantification of adenosine 5′-triphosphate (ATP) in spermatozoa and oocytes. The optimization of the precapillary derivatization reaction between ATP and 4,4-difluoro-5,7-dimethyl-4-bora-3a,4adiaza-s-indacene-3-propionyl ethylene diamine hydrochloride (BODIPY FL EDA) has been described. BODIPY-ATP conjugate was analysed in an uncoated fused silica capillary of 75 μm ID and 50 cm effective length using a 10 mmol/L tribasic sodium phosphate buffer, pH 11.5, at 22 kV in <5 min. A good reproducibility of intra- and inter-assay tests was obtained (CV?=?4.55% and 7.14%, respectively). With respect to our previous CE-UV assay, the new method showed an improvement in sensitivity that was about 120-fold (limit of quantification, 0.15 vs 18 μmol/L). Method applicability was proven on the reproductive cells of several animal species (roosters, horses, sheep and goats). Due to the elevated sensitivity, the new assay allows the measurement of adenosine 5′-triphosphate levels from just 20 oocytes. Considering that ATP concentration in reproductive cells is related to the mitochondrial integrity after cryopreservation, the proposed method could be a useful tool in assisted reproductive technologies.     

Ultrasensitive fluorescence protein detection in isoelectric focusing gels using a ruthenium metal chelate stain

SYPRO Ruby IEF Protein Gel Stain is an ultrasensitive, luminescent stain optimized for the analysis of protein in isoelectric focusing gels. Proteins are stained in a ruthenium-containing metal complex overnight and then rinsed in distilled water for 2 h. Stained proteins can be excited by ultraviolet light of about 302 nm (UV-B transilluminator) or with visible light of about 470 nm. Fluorescence emission of the dye is maximal at approximately 610 nm. The sensitivity of the SYPRO Ruby IEF protein gel stain is superior to colloidal Coomassie blue stain and the highest sensitivity silver staining procedures available. The SYPRO Ruby IEF protein gel stain is suitable for staining proteins in nondenaturing or denaturing carrier ampholyte isoelectric focusing and immobilized pH gradient gel electrophoresis. The stain is compatible with N,N'-methylenebisacrylamide or piperazine diacylamide cross-linked polyacrylamide gels as well as with agarose gels and high tensile strength Duracryl gels. The stain does not contain extraneous chemicals (formaldehyde, glutaraldehyde, Tween-20) that frequently interfere with peptide identification in mass spectrometry. Successful identification of stained proteins by peptide mass profiling is demonstrated.     

Capillary electrophoresis with laser-induced fluorescence detection as a tool for enzyme characterization and inhibitor screening.

An effective, rapid and economical CE/LIF (capillary electrophoresis/laser-induced fluorescence) method was developed and applied to the characterization of signal peptidase (SPase) enzyme, which is a target for the screening of new drug candidates. In this method, CE separates the product from the substrate and LIF selectively detects the fluorescence-labeled product and substrate. By measuring the increase of the product as a function of time, one can monitor the progression of the enzyme reaction. The progression curves were also used for screening inhibitors for this enzyme. The effects of various reaction conditions were also studied and discussed. In addition, this CE/LIF method was applied to the determination of the enzyme activity, the quality control of the substrate and/or enzymes, and the cross-reactivity of inhibitors to the enzyme. It can be concluded that this method is suitable for high throughput screening (HTS) assays because it can deliver fast, sensitive, quantitative, and reliable results.     

毛细管电泳-激光诱导荧光法测定草甘膦、草胺膦及氨甲基膦酸

建立了一种快速、有效的毛细管电泳分离-激光诱导荧光检测有机磷除草剂草甘膦、草胺膦和草甘膦的代谢物氨甲基膦酸的方法。将荧光衍生试剂5-(4, 6-二氯三嗪基)氨基荧光素(DTAF)成功用于衍生上述3种化合物。最佳衍生条件: DTAF的浓度为1.0 μmol/L,以50 mmol/L硼酸(pH 9.5)作为缓冲溶液,在30 ℃下反应40 min。以pH 9.5的30 mmol/L硼酸缓冲溶液(含15 mmol/L Brij-35)作为电泳背景电解质,3种衍生物得到基线分离。在优化的条件下,草甘膦、草胺膦、氨甲基膦酸的检出限分别为3.21、6.14和1.99 ng/kg。将该方法应用于环境水样和土壤中除草剂及代谢物的测定,回收率为91.3%～106.0%。该方法准确、灵敏,可满足环境样品中有机磷农药及其代谢物残留的检测要求。     

Application of capillary isoelectric focusing and peptide mass fingerprinting in carbohydrate-deficient transferrin detection

Carbohydrate-deficient transferrin (CDT) is a specific biomarker of alcohol abuse and is widely used in clinical diagnosis to detect and follow up excessive alcohol consumption. However, false %CDT results still exist in CDT detection, because of interference from genetic variants and the lack of standardization in CDT analysis. Therefore, it is still very important to find a method with high sensitivity and high accuracy for CDT detection. Here, we compared the detection sensitivity and accuracy of pI values based methods [isoelectric focusing on polyacrylamide gel electrophoresis (IEF-PAGE) and capillary isoelectric focusing (CIEF)] with hydrophobic characteristic based methods [reversed-phase high-performance liquid chromatography (RP-HPLC)] on CDT detection. Moreover, we investigated the potential of peptide mass fingerprinting (PMF), a method based on the mass spectrometry to identify human transferrin (HTf) variants including CDT isoforms and genetic variants, based on their specific peptide masses. Results indicated that PMF can identify HTf variants including CDT isoforms and genetic variants based on their specific peptides, and CIEF showed higher sensitivity detection of HTf variants than RP-HPLC and IEF-PAGE did. Accordingly, we suggest that PMF is suitable for identifying CDT with high accuracy, and CIEF has potential for detection of CDT and genetic variants with high sensitivity; moreover, they are both worth further investigation in clinical diagnosis.     

Capillary electrophoresis with laser-induced fluorescence detection of main polyamines and precursor amino acids in saliva

A hollow-fiber liquid-phase microextraction （HF-LPME） method has been developed for the purification and preconcentration of biogenic polyamines and their precursor amino acids in human saliva. Putrescine （Put）, cadaverine （Cad）, spermidine （Spe）, ornithine （Orn）, lysine （Lys）, and arginine （Arg） were determined by the CE-LIF detection after microextraction. Several factors that affect extraction efficiency, separation, and detection were investigated. Under the optimum conditions, six analytes could achieve baseline separation within 30 min, exhibiting a linear calibration at three orders of magnitude （r2 〉 0.998）; the obtained enrichment factors of HF-LPME were between 19 （for Orn） and 2] 8 （for Cad）, and the LODs were in the range of 0.0072-0.26 nmol/L. The proposed HF-LPME/CE-LIF method has been successfully applied for the sensitive analyses of the real-world saliva samples collected from healthy volunteers and different patients with oral diseases, providing a potential method for primary non-invasive diagnosis of some oral diseases.     

Dynamic pH junction-sweeping for on-line focusing of dipeptides in capillary electrophoresis with laser-induced fluorescence detection

In this paper, we developed a simple and effective on-line focusing technique combining dynamic pH junction and sweeping by capillary electrophoresis (CE) with laser-induced fluorescence (LIF) detection. Dynamic pH junction-sweeping is defined when the sample has a different buffer pH (dynamic pH junction condition) and is devoid of micelles (sweeping condition) relative to the background electrolyte (BGE). This hyphenated focusing mode was applied to the sensitive and selective focusing of four dipeptides: Tyr-Phe, Tyr-Leu, Trp-Gly, and Ala-Gln. Picomolar detectability of these dipeptides by CE-LIF detection was demonstrated through effective focusing of large sample volumes (up to 39% capillary length) using the dual pH junction-sweeping focusing mode. 25 mmol L(-1) sodium dihydrogen phosphate, pH 2.5 was used as the sample matrix, and 100 mmol L(-1) borate, 21 mmol L(-1) sodium dodecylsulfate (SDS), 16 mmol L(-1) Brij35, pH 9.0 as the background solution (BGS). The concentration detection limits (S/N = 3) of the four dipeptides were in the range of 1.0-5.0 pmol L(-1). The developed method has been successfully used for the determination of dipeptides in human serum samples.     

A DNA assay protocol in a lab-on-valve meso-fluidic system with detection by laser-induced fluorescence

An automatic protocol for in-situ assay of dsDNA is presented by employing a micro-sequential injection lab-on-valve meso-fluidic system, which facilitates precise fluidic handling at the 0.1-10 microl level. Sub-nano-liter to a few micro-liters of DNA sample and ethidium bromide (EB) solutions were introduced into the meso-fluidic system, where EB binding onto DNA takes place and an intercalated DNA-EB adduct was formed, which was afterwards excited in the flow cell of the LOV by a 473 nm laser beam, and the emitted fluorescence was monitored in-situvia optical fibers. The experimental variables, i.e., pH of the buffer solution, the concentration and volume of EB solution, the reaction time and the fluid flow rates, were investigated. By loading 600 nl sample and 1.0 microl EB solution, a linear calibration graph was obtained within 0.03-3.0 microg ml(-1)(dsDNA), and a detection limit (3sigma) of 0.009 microg ml(-1) was achieved, along with a sampling frequency of 60 h(-1) and a precision of 1.9% at the 1.0 microg ml(-1) level. The detection limit was further improved to 0.006 microg ml(-1) by increasing the sample volume to 2.0 microl. Plasmid DNA in E. Coli extraction and lambda-DNA/Hind III in four synthetic samples were assayed by using this procedure. For the plasmid DNA, a good agreement with the documented UV method was obtained, while spiking recoveries for the synthetic samples were 95.6-103.4%.     

Analysis of subcellular sized particles. Capillary electrophoresis with post-column laser-induced fluorescence detection versus flow cytometry

Flow cytometry (FCM) and more recently capillary electrophoresis with post-column laser-induced fluorescence detection (CE-LIF) have both been used for subcellular particle analysis but their analytical performance has not been compared. In this work, we compare a commercial FCM with an in-house built CE-LIF instrument using fluorescently labeled microspheres and isolated mitochondria. As evidenced by the relative standard deviation (RSD) of the individual fluorescence intensities, FCM is two-fold better than CE-LIF for microspheres with > or =1.5 x 10(6) molecules of equivalent soluble fluorescein (MESF). However, FCM has a comparatively low signal-to-noise ratio (S/N) and high RSD for microspheres with <1.5 x 10(6) MESF. CE-LIF, on the other hand, produces S/N ratios that are >25 times higher than FCM for all the microspheres tested and a lower RSD for microspheres with <1.5 x 10(6) MESF. When 10-N-nonyl acridine orange (NAO)-labeled mitochondria are analyzed, the S/N ratios of both techniques are similar. This appears to result from photobleaching of NAO-labeled mitochondria as they are detected by the LIF detector of the CE-LIF instrument. Both techniques have a niche in subcellular analysis; FCM has the advantage of collecting data for thousands of particles quickly, whereas CE-LIF consumes less than a nanoliter of sample and provides the electrophoretic mobility for individual particles.