Determination of micro‐RNA in cardiomyoblast cells using CE with LIF detection

Micro‐RNAs (miRNAs) are small, endogenous, singlestranded, and noncoding RNAs. The miRNAs have been found to perform important functions in many cellular processes, such as development, proliferation, differentiation, and apoptosis. Circulating miRNAs have been proposed as emerging biomarkers in diseases such as cancer, diabetes, and cardiovascular disease including acute myocardial infarction (AMI). In this study, we developed CE with LIF (CE‐LIF) using fluorescence‐labeled DNA probe for determination of low abundance miRNA in cell extracts. The target miRNA is miRNA‐499, a biomarker candidate of AMI with low abundance in biological samples. In order to measure the trace level of miRNA, we optimized the hybridization conditions such as hybridization time, temperature, and buffer solution. The highest fluorescence intensity of the hybridized miRNA‐499 was found when hybridization was conducted at 40°C in 50 mM Tris‐acetate (pH 8.0) buffer containing 50 mM NaCl, and 10 mM EDTA for 15 min. The hybridized miRNA‐499 was detected in cultured H9c2 cardiomyoblast cells and the analysis of miRNA‐499 was completed within 1 h using CE‐LIF. These results showed the potential of CE for fast, specific, and sensitive high‐throughput analysis of low‐abundance miRNAs in cell extracts, biofluids, and tissues.     

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.     

A PDMS sheath flow cuvette for high‐sensitivity LIF measurements in CE

A simple method for producing a sheath flow cuvette in PDMS suitable for post‐column detection in CE is described. Two types of cuvette were investigated. In the first, the sheath flow channel had a round cross‐section of approximately 635 μm diameter, whereas the second cuvette had a 300×300 μm² square channel. Both cuvettes produced laminar flows that ensheathed the separation capillary's effluent allowing sensitive fluorescence measurements. The elasticity of the PDMS allowed the 300×300 μm² square sheath flow channel to expand uniformly and accommodate the larger 330–340 μm od round separation capillary, producing a self‐aligning cuvette with robust mechanical properties. With this cuvette, linear calibrations of over five orders of magnitude and 15–30 zmol fluorescein detection limits were obtained for 12 and 50 μm id capillaries.     

Development of an SDS‐gel electrophoresis method on SU‐8 microchips for protein separation with LIF detection: Application to the analysis of whey proteins

This work describes the development of an SDS‐gel electrophoresis method for the analysis of major whey proteins (α‐lactalbumin, β‐lactoglobulin, and BSA) carried out in SU‐8 microchips. The method uses a low‐viscosity solution of dextran as a sieving polymer. A commercial coating agent (EOTrol LN) was added to the separation buffer to control the EOF of the chips. The potential of this coating agent to prevent protein adsorption on the walls of the SU‐8 channels was also evaluated. Additionally, the fluorescence background of the SU‐8 material was studied to improve the sensitivity of the method. By selecting an excitation wavelength of 532 nm at which the background fluorescence remains low and by replacing the mercury arc lamp by a laser in the detection system, an LOD in the nanomolar range was achieved for proteins derivatized with the fluorogenic reagent Chromeo P540. Finally, the method was applied to the analysis of milk samples, demonstrating the potential of SU‐8 microchips for the analysis of proteins in complex food samples.     

Light-emitting diode-induced fluorescence detection of native proteins in capillary electrophoresis

A continuous-wave 280 nm light-emitting diode (LED) was used as the excitation source for native fluorescence detection of proteins in CE. The operating current and temperature of the LED were optimized in order to achieve high luminescence power. It was found that a forward current of 30 mA and a temperature of approximately 5 degrees C gave the best S/N. By using a set of two ball lenses to focus light from the LED, we achieved a spot of approximately 200 mum with a power of 0.1-0.2 mW on the detection window. Fluorescence was collected with a ball lens at 90 degrees angle through a bandpass filter onto a photomultiplier tube. In CZE an LOD of 20 nM for conalbumin was reached. In capillary gel electrophoresis all eight proteins from a commercial standard kit were detected with high S/N. For a 10 microg/mL total protein mixture, S/N was better than 3 for all proteins in solution. Further improvement in LOD should be possible on utilization of an LED with higher luminescence power.     

A high‐sensitivity LIF detector with silver mirror coating detection window and small‐angle optical deflection from collinear system for CE

An LIF detector was integrated into a CE system based on silver mirror coating detection window and small‐angle optical deflection from collinear configuration. For this detection scheme, the incident light beam was focused on capillary through the edge of a lens, resulting in a small deflection angle that deviated 18° from the collinear configuration. Meanwhile, the excitation light and emitted fluorescence were effectively reflected by silver mirror coating at the detection window. The fluorescence was collected through the center of the same lens and delivered to a PMT in the vertical direction. In contrast to conventional collinear LIF detection systems, the fluorescence intensity was greatly enhanced and the background level was significantly eliminated. FITC and FITC‐labeled amino acids were used as model analytes to evaluate the performance with respect to design factors of this system. The limit LOD was estimated to be 0.5 pM for FITC (S/N = 3), which is comparable to that of optimized confocal LIF systems. All the results indicate that the proposed detection scheme will be promising for development of sensitive and low‐cost CE system.     

Heart‐cutting 2D‐CE with on‐line preconcentration for the chiral analysis of native amino acids

The use of transient moving chemical reaction boundary (tMCRB) was investigated for the on‐line preconcentration of native amino acids in heart‐cutting 2D‐CE with multiple detection points using contactless conductivity detection. The tMCRB focusing was obtained by using ammonium formate (pH 8.56) as sample matrix and acetic acid (pH 2.3) as a BGE in the first dimension of the heart‐cutting 2D‐CE. Different experimental parameters such as the injected volume and the concentration in ammonium formate were optimized for improving the sensitivity of detection. A stacked fraction from the first dimension was selected, isolated in the capillary, and then separated in the second dimension in the presence of a chiral selector ((+)‐(18‐crown‐6)‐2,3,11,12‐tetracarboxylic acid). This on‐line tMCRB preconcentration coupled with heart‐cutting 2D‐CE was applied with success to the chiral separation of D ,L ‐phenylalanine, and D ,L ‐threonine in a mixture of 22 native amino acids. The sample mixture was diluted in 0.8 M of ammonium formate, and injected at a concentration of 2.5 μM for each enantiomer with a volume corresponding to 10% of the total capillary volume. An LOD (S/N=3) of 2 μM was determined for L ‐threonine.     

Analysis of pharmaceutical impurities using multi‐heartcutting 2D LC coupled with UV‐charged aerosol MS detection

To overcome challenges in HPLC impurity analysis of pharmaceuticals, we developed an automated online multi‐heartcutting 2D HPLC system with hyphenated UV‐charged aerosol MS detection. The first dimension has a primary column and the second dimension has six orthogonal columns to enhance flexibility and selectivity. The two dimensions were interfaced by a pair of switching valves equipped with six trapping loops that allow multi‐heartcutting of peaks of interest in the first dimension and also allow “peak parking.” The hyphenated UV‐charged aerosol MS detection provides comprehensive detection for compounds with and without UV chromophores, organics, and inorganics. It also provides structural information for impurity identification. A hidden degradation product that co‐eluted with the drug main peak was revealed by RP × RP separation and thus enabled the stability‐indicating method development. A poorly retained polar component with no UV chromophores was analyzed by RP × hydrophilic interaction liquid chromatography separation with charged aerosol detection. Furthermore, using this system, the structures of low‐level impurities separated by a method using nonvolatile phosphate buffer were identified and tracked by MS in the second dimension.     

Capacitively coupled contactless conductivity detection as an alternative detection mode in CE for the analysis of kanamycin sulphate and its related substances

A method was developed to determine simultaneously kanamycin, its related substances and sulphate in kanamycin sulphate using capacitively coupled contactless conductivity detection. Kanamycin is an aminoglycoside antibiotic that lacks a strong UV-absorbing chromophore. Due to its physicochemical properties, CE in combination with capacitively coupled contactless conductivity detection was chosen. The separation method uses a BGE composed of 40 mM 2-(N-morpholino)ethanesulphonic acid monohydrate and 40 mM L-histidine, pH 6.35. A 0.6 mM N-cetyltrimethyl ammonium bromide (CTAB) solution was added as electroosmotic flow modifier in a concentration below the critical micellar concentration (CMC). Ammonium acetate 50 mg/L was used as internal standard. In total, 30 kV was applied in reverse polarity on a fused-silica capillary (65/41 cm; 75 μm id). The optimized separation was obtained in less than 6 min with good linearity (R(2)=0.9999) for kanamycin. It shows a good precision expressed as RSD on the relative peak areas equal to 0.3 and 1.1% for intra-day and inter-day precision, respectively. The LOD and LOQ are 0.7 and 2.3 mg/L, respectively. Similarly, for sulphate, a good linearity (R(2)=0.9996) and precision (RSD 0.4 and 0.6% for intra-day and inter-day, respectively) were obtained.     

Total nucleotide analysis of Hydra DNA and RNA by MEKC with LIF detection and 32P‐postlabeling

The model organism Hydra has been used for molecular studies for more than 20 years, however, its DNA base composition has not been determined yet. We have analyzed DNA and total RNA of the freshwater polyp Hydra magnipapillata with two independent procedures of high accuracy and sensitivity – fluorescence labeling of nucleotides followed by CE‐LIF detection and ³²P‐postlabeling. DNA of Hydra was digested either to deoxyribonucleoside‐5′‐monophosphates or deoxyribonucleoside‐3′‐monophosphates selectively conjugated with the fluorescent dye 4,4‐difluoro‐5,7‐dimethyl‐4‐bora‐3a,4a‐diaza‐s‐indacene‐3‐propionyl ethylene diamine hydrochloride (BODIPY FL EDA) separated and detected using CE‐LIF. Both versions of the assay revealed a high A+T composition of 78 and 71%, whereas total DNA methylation (5‐methyldeoxycytidine) was 2.6 and 3.1%. Total Hydra RNA showed highest base levels for guanine (33%) and a level of 1.4% for pseudouracil. All values were in good agreement with those determined by the ³²P‐postlabeling method.     

Boron‐chelating fluorescent probe (BOPB) in the red region combined with CE‐LIF for the detection of NO in mice liver

Precise measurement of nitric oxide (NO) is of great importance to understand the function of NO in liver and the mechanism of liver injury. 8‐(3’,4’‐Diamino phenyl)‐3,5‐(2‐hydroxyphenyl)‐dimethylene pyrrole (BOPB), a fluorescent probe in the red region (>600 nm) newly developed in our group, has good photostability and excitation/emission wavelength of 622/643 nm matching well with commercial 635 nm semiconductor laser of CE‐LIF detection. Therefore, BOPB was used in CE‐LIF for the determination of NO in mice liver. Both derivatization and separation conditions were optimized. Derivatization reaction of BOPB and NO was carried out in pH 7.4 PBS buffer at 35°C for 12 min and the separation of NO derivative of BOPB (BOPB‐T) was achieved within 7.0 min in pH 9.0 running buffer containing 15 mM H₃BO₃–NaOH and 15 mM SDS. Good linearity was found in the range of 1.0 × 10^?9–5.0 × 10^?7 M with the LOD of 0.02 nM. The proposed method was applied to the analysis of NO in real samples, and NO concentration was obviously increased in acute liver injury of mice. Compared to existing derivatization‐based CE‐LIF methods for NO, this method has lower LOD and less background interference owing to detection wavelength of BOPB in the red region.     

A comparative analysis of human plasma and serum proteins by combining native PAGE,whole‐gel slicing and quantitative LC‐MS/MS: Utilizing native MS‐electropherograms in proteomic analysis for discovering structure and interaction‐correlated differences

MS identification has long been used for PAGE‐separated protein bands, but global and systematic quantitation utilizing MS after PAGE has remained rare and not been reported for native PAGE. Here we reported on a new method combining native PAGE, whole‐gel slicing and quantitative LC‐MS/MS, aiming at comparative analysis on not only abundance, but also structures and interactions of proteins. A pair of human plasma and serum samples were used as test samples and separated on a native PAGE gel. Six lanes of each sample were cut, each lane was further sliced into thirty‐five 1.1 mm × 1.1 mm squares and all the squares were subjected to standardized procedures of in‐gel digestion and quantitative LC‐MS/MS. The results comprised 958 data rows that each contained abundance values of a protein detected in one square in eleven gel lanes (one plasma lane excluded). The data were evaluated to have satisfactory reproducibility of assignment and quantitation. Totally 315 proteins were assigned, with each protein assigned in 1–28 squares. The abundance distributions in the plasma and serum gel lanes were reconstructed for each protein, named as “native MS‐electropherograms”. Comparison of the electropherograms revealed significant plasma‐versus‐serum differences on 33 proteins in 87 squares (fold difference > 2 or < 0.5, p < 0.05). Many of the differences matched with accumulated knowledge on protein interactions and proteolysis involved in blood coagulation, complement and wound healing processes. We expect this method would be useful to provide more comprehensive information in comparative proteomic analysis, on both quantities and structures/interactions.     

A CE‐LIF method based on long wavelength fluorescence labeling for the analysis of thiols in human urine

A rapid and robust CE method using a long wavelength fluorescent reagent 1,7‐dimethyl‐3,5‐distyryl‐8‐phenyl‐(2‐maleimide)difluoroboradiaza‐s‐indacene as the labeling reagent has been developed for the simultaneous determination of thiols, including glutathione, cysteine, homocysteine, N‐acetylcysteine, cysteinylglycine, and penicillamine. The derivatization reaction was carried out in 14 mmol/L pH 8.5 borate buffer at 30°C for 6 min and the labeled thiols derivatives were separated with the running buffer containing 30 mmol/L pH 7.4 phosphate, 30% v/v acetonitrile and 8 mmol/L SDS within 12 min. Detection limits ranged from 0.4 to 2.4 nmol/L. To demonstrate the capability of this method, it was applied to the analysis of thiols in human urine with recoveries of 92.4–105.6%. The derivatization reaction was much faster at milder conditions, and the analysis was rapider. Moreover, with excitation wavelength at long wavelength region, background interference from samples was reduced effectively. The present method seems to be a potential choice for quantifying thiols in human urine.     

Fast screening of aflatoxins in dairy cattle feeds with CE‐LIF method combined with preconcentration technique of vortex assisted low density solvent–microextraction

Aflatoxin contamination in agricultural products poses a great threat to humans and livestock. The aim of this study was to establish a simple, rapid, highly sensitive, and inexpensive method for the simultaneous detection of aflatoxin B₁, B₂, G₁, and G₂ in agricultural products. We used a vortex assisted low density solvent–microextraction (VALDS‐ME) technique for sample preconcentration and sample detection was achieved with a CE‐LIF method. Aflatoxins were separated in an uncoated fused‐silica capillary with the MEKC mode and were excited by a 355 nm UV laser to produce native fluorescence for detection. The obtained LOD and LOQ for the four aflatoxins were in the range of 0.002–0.075 and 0.007–0.300 μg/L, respectively, and the analysis time was within 6.5 min. Using the established method, aflatoxins were screened in naturally contaminated dairy cattle feed samples including alfalfa, bran, and corn kernel. The result shows that the alfalfa and bran samples were contaminated with aflatoxins to varying degrees. Compared with other analytical techniques for aflatoxin screening in agricultural products, this CE‐LIF method combined with VALDS‐ME preconcentration technique is simple, rapid, highly efficient, and inexpensive.     

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.     

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.     

Global mapping of rat plasma proteins with a native proteomic approach using nondenaturing micro 2DE and quantitative LC‐MS/MS

Plasma samples from adult male rats were separated by nondenaturing micro 2DE and a reference gel was selected, on which 136 CBB‐stained spots were numbered and subjected to in‐gel digestion and quantitative LC‐MS/MS. The analysis provided the assignment of 1–25 (average eight) non‐redundant proteins in each spot and totally 199 proteins were assigned in the 136 spots. About 40% of the proteins were detected in more than one spot and 15% in more than ten spots. We speculate this complexity arose from multiple causes, including protein heterogeneity, overlapping of protein locations and formation of protein complexes. Consequently, such results could not be appropriately presented as a conventional 2DE map, i.e. a list or a gel pattern with one or a few proteins annotated to each spot. Therefore, the LC‐MS/MS quantity data was used to reconstruct the gel distribution of each protein and a library containing 199 native protein maps was established for rat plasma. Since proteins that formed a complex would migrate together during the nondenaturing 2DE and thus show similar gel distributions, correlation analysis was attempted for similarity comparison between the maps. The protein pairs showing high correlation coefficients included some well‐known complexes, suggesting the promising application of native protein mapping for interaction analysis. With the importance of rat as the most commonly used laboratory animal in biomedical research, we expect this work would facilitate relevant studies by providing not only a reference library of rat plasma protein maps but a means for functional and interaction analysis.     

Quantitative determination and evaluation of Paris polyphylla var. yunnanensis with different harvesting times using UPLC‐UV‐MS and FT‐IR spectroscopy in combination with partial least squares discriminant analysis

A rapid method was developed and validated by ultra‐performance liquid chromatography–triple quadrupole mass spectroscopy with ultraviolet detection (UPLC‐UV‐MS) for simultaneous determination of paris saponin I, paris saponin II, paris saponin VI and paris saponin VII. Partial least squares discriminant analysis (PLS‐DA) based on UPLC and Fourier transform infrared (FT‐IR) spectroscopy was employed to evaluate Paris polyphylla var. yunnanensis (PPY) at different harvesting times. Quantitative determination implied that the various contents of bioactive compounds with different harvesting times may lead to different pharmacological effects; the average content of total saponins for PPY harvested at 8 years was higher than that from other samples. The PLS‐DA of FT‐IR spectra had a better performance than that of UPLC for discrimination of PPY from different harvesting times.     

Investigation of commercial sorbents for the analysis of opioid peptides in human plasma by on‐line SPE‐CE

In this study, we investigated the performance of several commercial sorbents (Sep‐pack^® C18, ^tC18, C8 and ^tC2, Oasis^® HLB, Isolute^® ENV+, Strata^?‐X and Oasis^® MCX) for the determination of opioid peptides by solid‐phase extraction coupled on‐line to capillary electrophoresis (SPE‐CE). First, standard solutions were analyzed in order to achieve the lowest LOD and the best electrophoretic separations using UV detection. The best results were obtained using C18, C8 and ^tC2 sorbents, which were examined for the analysis of spiked human plasma samples. A double‐step sample clean‐up pretreatment, which consisted of precipitation with acetonitrile and filtration, was needed to prevent saturation of the on‐line SPE microcartridge. The filtration step was critical to obtain optimum analyte recovery and to clean up the sample matrix. A range of centrifugal filters and filtration conditions were tested and the recoveries of the sample pretreatment were evaluated by CE‐ESI‐MS. The LODs attained through SPE‐CE‐UV were approximately ten‐fold better with C18 than with C8 and ^tC2. The 0.1 μg/mL LODs achieved by C18‐SPE‐CE‐UV were further improved until we could detect 1 ng/mL concentrations of opioid peptides in plasma samples by C18‐SPE‐CE‐ESI‐MS, due to the outstanding selectivity of the MS detection.     

Analysis of low‐density lipoprotein‐associated proteins using the method of digitized native protein mapping

The method of digitized native protein mapping, combining nondenaturing micro 2DE, grid gel‐cutting, and quantitative LC‐MS/MS (in data‐independent acquisition mode, or MS^E), was improved by using a new MS/MS mode, ion mobility separation enhanced‐MS^E (HDMS^E), and applied to analyze the area of human plasma low‐density lipoprotein (LDL). An 18 mm × 4.8 mm rectangular area which included LDL on a nondenaturing micro 2D gel of human plasma was grid‐cut into 72 square gel pieces and subjected to quantitative LC‐MS/MS. Compared with MS^E, HDMS^E showed significantly higher performance, by assigning 50% more proteins and detecting each protein in more squares. A total of 253 proteins were assigned with LC‐HDMS^E and the quantity distribution of each was reconstructed as a native protein map. The maps showed that Apo B‐100 was the most abundant protein in the grid‐cut area, concentrated at pI ca. 5.4–6.1 and apparent mass ca. 1000 kDa, which corresponded to four gel pieces, squares 39–42. An Excel macro was prepared to search protein maps which showed protein quantity peaks localized within this concentrated region of Apo B‐100. Twenty‐two proteins out of the 252 matched this criterion, in which 19 proteins have been reported to be associated with LDL. This method only requires several microliters of a plasma sample and the principle of the protein separation is totally different from the commonly used ultracentrifugation. The results obtained by this method would provide new insights on the structure and function of LDL.