Capillary sodium dodecyl sulfate-DALT electrophoresis of proteins in a single human cancer cell.

Capillary Sodium dodlecyl sulfate (SDS)-DALT an (abbreviation for Dalton) electrophoresis was applied to analysis of proteins in single HT29 human colon adenocarcinoma cells. A vacuum pulse was employed to introduce a single cell into the coated capillary. Once the cell was lysed, proteins were denatured with SDS, fluorescantly labeled with 3-(2-furoyl)-quinoline-2-carboxaldehyde (FQ), and then separated by using 8% pullulan as the sieving matrix. This method offers a few advantages for single-cell protein analysis. First, it provides reproducible separation of single-cell proteins according to their size. Based on comparison with the migration time of standard proteins, most components from a single HT29 cancer cell have molecular masses within the range of 10-100 kDa. Second, as a one-dimensional separation method, it gives fairly good resolution for proteins. Typically, around 30 protein components of a single HT29 cell were resolved, indicating that this method has similar peak capacity to SDS-polyacrylamide gel electrophoresis (PAGE). Third, this method shows high detection sensitivity and wide dynamic range, which is important because of the wide range of protein expression in living systems. Detection limits for standard proteins ranged from 10(-10) to 10(-11) M. Finally, this method provides much higher speed than classical gel electrophoresis methods, and it provides automated anlysis of cellular proteins at the single-cell level; the separation is complete in 30 min and the entire analysis takes approximately 45 min.

Biomonitoring of urinary tamoxifen and its metabolites from breast cancer patients using nonaqueous capillary electrophoresis with electrospray mass spectrometry.

Tamoxifen is an antiestrogen drug used to treat breast cancer. We have extracted tamoxifen and several of its metabolites from urine of patients with both metastatic (stage IV) and locally confined (stages I, II, and III) breast cancer. Analysis of these metabolites was performed by nonaqueous capillary electrophoresis with electrospray-mass spectrometry. Peak heights from extracted ion current electropherograms of the metabolites were used to establish a metabolic profile for each patient. We demonstrate substantial variation among patient profiles, statistically significant differences in the amount of urinary tamoxifen N-oxide found in stages I, II, and III compared to stage IV breast cancer patients, and statistically significant differences in the amount of 3,4-dihydroxytamoxifen found in progressors compared to nonprogressors with metastatic (stage IV) cancer.

Characterization of the interaction between phospholipid and protein by capillary electrophoresis with laser-induced fluorescence detection.

We report an electrophoretic mobility shift-based method to study the interactions between phospholipids and proteins by capillary electrophoresis with laser-induced fluorescence detection. A fluorogenic dye, 3-(2-furoyl)quinoline-2-carboxaldehyde (FQ), was used to label phosphatidylserine (PS). Then the FQ labeled PS (FQ-PS) was used as the fluorescent probe for monitoring the association between PS and bovine serum albumin (BSA). Two conjugates were observed to form between each PS species and BSA, indicating that two interactions exist between these PS species and BSA. We can also detect the competitive association with BSA between labeled PS and unlabeled PS. This method only needs a minute volume of sample. It is highly sensitive and can be used to detect the interaction between phospholipids and nanomolar concentrations of proteins, such as BSA.

Manipulation of protein fingerprints during on-column fluorescent labeling: protein fingerprinting of six Staphylococcus species by capillary electrophoresis.

Bacterial proteomes were analyzed by use of electrophoretically mediated microanalysis (EMMA) and field-enhanced stacking. A water-soluble protein fraction was injected onto a capillary. Next, a fluorogenic reagent was injected and allowed to react with the protein mixture, producing fluorescent products that were separated by submicellar capillary electrophoresis and detected by laser-induced fluorescence. By use of a low-ionic strength sample buffer and a brief electrophoretic step, slow moving anionic proteins were stacked at the reagent-sample interface and were preferentially labeled. By reversing the order of sample injection and labeling reagent, fast moving cationic proteins were preferentially labeled. By adjustment of the sample buffer pH, proteins with different isoelectric points were selectively labeled. Electrophoresis fingerprints were generated for the water-soluble protein fraction from six Staphylococcus species. The protein patterns produced were species-specific and were used to construct a phylogenetic tree.

Application of capillary electrophoresis with laser-induced fluorescence detection to the determination of biogenic amines and amino acids in brain microdialysate and homogenate samples.

A procedure is described to derivatize 16 primary-amine-containing biogenic amines and amino acids in brain mixtures with the fluorogenic reagent 5-furoylquinoline-3-carboxaldehyde (FQ). These FQ-tagged compounds in the brain sample were resolved in less than 16 min based on micellar electrokinetic chromatography and laser-induced fluorescence. There was a linear relationship between the concentration of analyte and the fluorescence intensity, with correlation coefficients in the range of 0.96-1.00. The utility of this method for the quantification of the important inhibitory neurotransmitter gamma-aminobutyric acid in microdialysates and brain homogenates from rats is illustrated.

Analysis of tamoxifen and its metabolites in synthetic gastric fluid digests and urine samples using high-performance liquid chromatography with electrospray mass spectrometry.

We report on the transformation of tamoxifen at 37 degrees C in synthetic gastric fluid as studied by high-performance liquid chromatography with triple quadrupole mass spectrometry. The major transformation products detected were (E)-isomer of tamoxifen, metabolite D, and several unidentified components having m/z 404. Addition of pepsin to the gastric fluid inhibited formation of all of these products. We analyzed several urine samples from breast cancer patients undergoing tamoxifen treatment. Metabolite D was identified in the urine samples and in the gastric fluid digest at a retention time of 22.0 min eluting from a reversed-phase HPLC column. Although several metabolites were found in all the urine samples of patients, some metabolites were detected in one sample but not others, suggesting tamoxifen metabolism varies in patients.

Two-dimensional direct-reading fluorescence spectrograph for DNA sequencing by capillary array electrophoresis.

We report a compact, two-dimensional direct-reading fluorescence spectrograph and demonstrate its application to DNA sequencing by capillary array electrophoresis. The detection cuvette is based on sheath flow, wherein the capillaries terminate in a two-dimensional array in a fluid-filled chamber that is pressurized with buffer. A thin metal plate is located downstream from the capillaries. This barrier plate has an array of holes that precisely matches the location of the capillaries. Buffer flows through the holes, drawing analyte from the capillaries in a well-defined array of thin filaments. Fluorescence is excited in the upper chamber with an elliptically shaped laser beam. The bottom chamber is sealed with a glass window and drained from the side. Fluorescence is detected by imaging the illuminated sample streams through the holes in the barrier plate. A prism is used to disperse fluorescence from each sample across a CCD camera so that the emission spectrum is monitored simultaneously from each capillary. The instrument is demonstrated in a 32-capillary configuration but can be scaled to several thousand capillaries.

The effect of temperature oscillations on DNA sequencing by capillary electrophoresis.

Although capillary electrophoresis is a powerful sequencing technology, the low heat capacity of a capillary can make difficult the precise control of its temperature, particularly when the capillary is heated to reduce compressions in the separation of DNA sequencing fragments. In this paper, we demonstrate that minute oscillations in the capillary's temperature result in significant degradation in the number of theoretical plates, the resolution between adjacent peaks, and the number of bases of DNA sequence determined from the electrophoresis data. Temperature must be held stable to within 0.1 degrees C to obtain long read lengths. A Monte Carlo simulation demonstrates that this degradation is consistent with laminar flow induced by the periodic thermal expansion and contraction of the separation medium.

Protein analysis of an individual Caenorhabditis elegans single-cell embryo by capillary electrophoresis.

We present a simple one-dimensional electrophoretic map of the expressed proteins in a Caenorhabditis elegans embryo. The embryo was taken from an adult nematode, injected into a 50-microm I.D. capillary, and lysed. The proteins were fluorescently labeled and then separated by capillary electrophoresis and detected by laser-induced fluorescence. Over 20 components were resolved in the 22-min separation. The dynamic range was outstanding for this separation, noise in the baseline was less than 0.01% the amplitude of the largest component.

Non-aqueous capillary electrophoresis of tamoxifen and its acid hydrolysis products.

Tamoxifen and its acid hydrolysis products were separated and tentatively identified by non-aqueous capillary electrophoresis with thermooptical absorbance and electrospray ionization mass spectrometry. Acid hydrolysis is a convenient method of generating tamoxifen degradation products. The parent compound and seven hydrolysis products were separated in 9 min.

Effects of amine modifiers on the separation of tetramethylrhodamine-labeled mono- and oligosaccharides by capillary zone electrophoresis.

In this work, nine tetramethylrhodamine (TMR) labeled isomeric oligosaccharide derivatives of betaGal(1 --> 4) betaGlcNAc-O-TMR were separated by capillary zone electrophoresis coupled with laser-induced fluorescence detection. Charged species were created in situ by complexation with borate and phenylborate. Micellar separation was achieved by addition of 10 mM sodium dodecylsulfate to the running buffer. We have investigated the effects of adding a homologous series of monoamine modifiers on the separation efficiency of these oligosaccharides. The separation was significantly improved in the presence of the organic modifiers methyl- and ethylamines, but worsened in the presence of propyl- and butylamines. Possible mechanisms of the amine additives are discussed.

Methyl-beta-cyclodextrin modified micellar electrokinetic capillary chromatography with laser-induced fluorescence for separation and detection of phospholipids.

Micellar electrokinetic capillary chromatography (MECC) with laser-induced fluorescence detection was applied to the separation of amino group-containing phospholipids including phosphatidylethanolamine (PE), phosphatidylserine (PS), lysophosphatidylethanolamine (LysoPE), and lysophosphatidylserine (LysoPS). A fluorogenic dye, 3-(2-furoyl)quinoline-2-carboxaldehyde (FQ), was successfully used to fluorescently label these phospholipids. 4-Fluoro-7-nitrobenzofurazan only produced fluorescent product from LysoPE and PE; signals were not observed from LysoPS and PS. A borax buffer containing sodium deoxycholate modified with methyl-beta-cyclodextrin (methyl-beta-CD) was an excellent MECC system for these phospholipids. Under the optimum conditions, four FQ-labeled phospholipid classes were separated within 8 min. Moreover, each of the PE, PS, LysoPE and LysoPS peaks split into two components corresponding to subclasses with different lengths of the fatty acid chains, but these subclasses were completely resolved only for LysoPE. Detection limits ranged from 0.18 to 1.1 fg (10(-9) - 10(-10) M), which was four- to five-orders of magnitude superior to previously reported CE methods.

Separation of proteins by sodium dodecylsulfate capillary electrophoresis in hydroxypropylcellulose sieving matrix with laser-induced fluorescence detection.

Sodium dodecyl sulfate capillary electrophoresis by using hydroxypropylcellulose as the sieving matrix was developed for separation of proteins. 3-(2-furoyl)quinoline-2-carboxaldehyde, a fluorogenic dye, was used as the pre-column reagent to label proteins, which allows the use of laser-induced fluorescence to improve the detection sensitivity. Five standard proteins within the molecular mass range of 14,000-97,000 were used to test this method and a calibration curve was obtained between the molecular mass of these proteins and their peak migration times. This method was also applied to the separation of proteins from HT29 human colon adenocarcinoma cell extracts, and, typically, nearly 30 protein components could be resolved in a 20-min separation. Similar separation patterns were observed for the cell extract proteins when three running buffer systems were employed, indicating that buffer composition did not have much influence on the separation based on HPC sieving.

Single-cell analysis avoids sample processing bias.

Microscale separation tools such as capillary chromatography and capillary electrophoresis (CE) allow the study of metabolism in individual cells. In this work, we demonstrate that single-cell analysis describes metabolism more accurately than analysis of cellular extracts. We incubated HT29 cells (human colon adenocarcinoma) with a fluorescently labeled metabolic probe. This disaccharide, LacNAc, was labeled with a fluorescent dye, tetramethylrhodamine (TMR). The probe was taken up by the cells and metabolized to a number of products that retained the fluorescent label. We then split the cells into two batches. A cellular extract was prepared from one batch and analyzed by CE with laser-induced fluorescence (LIF) detection. The cells from the second batch were used for single-cell analysis by CE-LIF. Separation and detection conditions were identical for extract and single-cell analyses. We found that the electropherogram obtained by averaging the results from a number of single cells differed significantly from the cell extract electropherogram. Differences were due to sample processing during extract preparation. Disruption of the cells liberated enzymes that were compartmentalized within the cell, which allowed non-metabolic reactions to proceed. The accumulation of these non-metabolic products introduced a bias in the cell extract assay. During single-cell analysis, cells were lysed inside the capillary and the separation voltage was applied immediately to separate the enzymes from their substrates and prevent non-metabolic reactions. This paper is the first to report that CE analysis of single cells provides more accurate metabolic information than the CE analysis of a cellular extract.

Reaction rate, activation energy, and detection limit for the reaction of 5-furoylquinoline-3-carboxaldehyde with neurotransmitters in artificial cerebrospinal fluid.

The fluorogenic reagent 5-furoylquinoline-3-carboxaldehyde (FQ) has proven valuable in the analysis of proteins and small neurotransmitters. We monitored the reaction rate between this reagent and five neurotransmitters at 40 degrees and 65 degrees C in artificial cerebrospinal fluid. The reactions followed pseudo-first order kinetics. The activation energy for the reaction of FQ was 10.6, 10.7, 22.0, 31.4, and 34.4 kJ mol(-1) for alanine, taurine, gamma-aminobutyric acid (GABA), glutamine, and glutamic acid, respectively. At 65 degrees C, the reaction rate was quite similar for alanine, taurine, glutamine, and glutamic acid (1.8 x 10(-3) s(-1)) but was twice as fast for GABA. A reaction time of nearly 1 h was required to quantitatively convert these neurotransmitters to their fluorescent products at 65 degrees C. Detection limits for the labeled neurotransmitters were 10(-9)-10(-8) M, which corresponded to 0.3-7 amol injected onto the capillary.

Construction and evaluation of a capillary array DNA sequencer based on a micromachined sheath-flow cuvette.

A capillary array electrophoresis DNA sequencer is reported based on a micromachined sheath-flow cuvette as the detection chamber. This cuvette is equipped with a set of micromachined features that hold the capillaries in precise registration to ensure uniform spacing between the capillaries, in order to generate uniform hydrodynamic flow in the cuvette. A laser beam excites all of the samples simultaneously, and a microscope objective images fluorescence onto a set of avalanche photodiodes, which operate in the analog mode. A high-gain transimpedance amplifier is used for each photodiode, providing high duty-cycle detection of fluorescence.