Norman J. Dovichi

High-sensitivity fluorescence detector for fluorescein isothiocyanate derivatives of amino acids separated by capillary zone electrophoresis

A fluorescence detector has been developed for capillary zone electrophoresis that produces a ten-fold improvement in precision compared with the previous state-of-the-art in fluorescence detection. This instrument, which is based on a sheath-flow cuvette flow chamber and a 0.05-W argon ion laser beam, combines a high numerical aperture collection optic, N.A = 0.65, with a high quantum yield photomultiplier tube, phi approximately = 0.15 at the wavelength of maximum emission. Detection limits (3 sigma) range from the injection of 1.7 . 10(-21) mol (1.3 . 10(-12) M) of fluorescein isothiocyanate (FITC)-labeled arginine, the best case, to 6 . 10(-21) mol (5.6 . 10(-12) M) of FITC-cysteine, the worse case. Signal linearity extends for at least five orders of magnitude from the detection limit to greater than 10(-16) mol (10(-7) M) injected.

Capillary gel electrophoresis for DNA sequencing : Laser-induced fluorescence detection with the sheath flow cuvette

Capillary polyacrylamide gel electrophoresis separation of dideoxycytidine chain-terminated DNA fragments is reported. A post-column laser-induced fluorescence detector based on the sheath flow cuvette was used to minimize background signals due to light scatter from the gel and capillary. A preliminary mass detection limit of 10(-20) mol of fluorescein-labeled DNA fragments was obtained. The system was used to analyze an actual DNA sequencing sample. Theoretical plate counts of 2 x 10(6) were produced. Gel stability limits the performance of the current system.

Simplified capillary electrophoresis nanospray sheath‐flow interface for high efficiency and sensitive peptide analysis

We report a simple nanospray sheath-flow interface for capillary electrophoresis. This interface relies on electrokinetic flow to drive both the separation and the electrospray; no mechanical pump is used for the sheath flow. This system was interfaced with an LCQ mass spectrometer. The best results were observed with a 2-microm diameter emitter tip and a 1-mm spacing between the separation capillary tip and the emitter tip. Under these conditions, mass detection limits (3sigma) of 100 amol were obtained for insulin receptor fragment 1142-1153. The separation efficiency exceeded 200,000 plates for this compound. The relative standard deviation generated during continual infusion of a 50 microM solution of angiotensin II was 2% for the total ion count and 3% for the extracted ion count over a 40-min period. Finally, the interface was also demonstrated for negative ion mode.

Species identification and phylogenetic relationships based on partial HSP60 gene sequences within the genus Staphylococcus

The phylogenetic relationships among 36 validly described species or subspecies within the genus Staphylococcus were investigated by cloning and sequencing their 60 kDa heat-shock protein (HSP60) genes using a set of universal degenerate HSP60 PCR primers. The cloned partial HSP60 DNA sequences from nine Staphylococcus aureus strains were highly conserved (97-100% DNA sequence similarity; mean 98%), indicating that the HSP60 gene of multiple isolates within the same species have little microheterogeneity. At the subspecies level, DNA sequence similarity among members of S. aureus, Staphylococcus schleiferi, Staphylococcus cohnii and Staphylococcus capitis ranged from 91 to 98%. At the interspecies level, sequence similarity among 23 distinct species of staphylococci ranged from 74 to 93% (mean 82%). By comparison, the highest sequence similarity of Bacillus subtilis and Escherichia coli with members within the genus Staphylococcus was only 70 and 59%, respectively. Importantly, phylogenetic analysis based on the neighbour-joining distance method revealed remarkable concordance between the tree derived from partial HSP60 gene sequences and that based on genomic DNA-DNA hybridization, while 16S rRNA gene sequences correlated less well. The results demonstrate that DNA sequences from the highly conserved and ubiquitous HSP60 gene offer a convenient and accurate tool for species-specific identification and phylogenetic analysis of staphylococci.

Low-cost, high-sensitivity laser-induced fluorescence detection for DNA sequencing by capillary gel electrophoresis

A low cost, 0.75-mW helium neon laser, operating in the green region at 534.5 nm, is used to excite fluorescence from tetramethylrhodamine isothiocyanate-labelled DNA fragments that have been separated by capillary gel electrophoresis. The detection limit (3 sigma) for the dye is 500 ymol [1 yoctomole (1 ymol) = 10(-24) mol] or 300 analyte molecules in capillary zone electrophoresis; the detection limit for labeled primer separated by capillary gel electrophoresis is 2 zmol [1 zeptomole (1 zmol) = 10(-21) mol]. The Richardson-Tabor peak-height encoded sequencing technique is used to prepare DNA sequencing samples. In 6% T, 5% C acrylamide, 7 M urea gels, sequencing rates of 300 bases/hour are produced at an electric field strength of 200 V/cm; unfortunately, the data are plagued by compressions. These compressions are eliminated with addition of 20% formamide to the sequencing gel; the gel runs slowly and sequencing data are generated at a rate of about 70 bases/hour.

Picomolar assay of native proteins by capillary electrophoresis precolumn labeling, submicellar separation, and laser-induced fluorescence detection.

We report a method for the assay of proteins at concentrations lower than 10(-)(10) M with as little as 200 amol of protein. High sensitivity is accomplished by derivatizing the ε-amino group of the proteins lysine residues with the fluorogenic dye 5-furoylquinoline-3-carboxaldehyde and use of a sheath flow cuvette fluorescence detector. Most proteins have a large number of lysine residues; therefore, a large number of fluorescent molecules can be attached to each protein molecule. In general, precolumn labeling improves sensitivity but degrades resolution due to the inhomogeneity of the reaction products from multiple labeling. However, we demonstrate that, through careful manipulation of the separation and reaction conditions, high sensitivity can be obtained without excessive loss in separation efficiency. Over 190 000 theoretical plates are obtained for fluorescently labeled ovalbumin.

Capillary Zone Electrophoresis-Electrospray Ionization-Tandem Mass Spectrometry as an Alternative Proteomics Platform to Ultraperformance Liquid Chromatography-Electrospray Ionization-Tandem Mass Spectrometry for Samples of Intermediate Complexity

We demonstrate the use of capillary zone electrophoresis with an electrokinetically pumped sheath-flow electrospray interface for the analysis of a tryptic digest of a sample of intermediate protein complexity, the secreted protein fraction of Mycobacterium marinum. For electrophoretic analysis, 11 fractions were generated from the sample using reverse-phase liquid chromatography; each fraction was analyzed by CZE-ESI-MS/MS, and 334 peptides corresponding to 140 proteins were identified in 165 min of mass spectrometer time at 95% confidence (FDR < 0.15%). In comparison, 388 peptides corresponding to 134 proteins were identified in 180 min of mass spectrometer time by triplicate UPLC-ESI-MS/MS analyses, each using 250 ng of the unfractionated peptide mixture, at 95% confidence (FDR < 0.15%). Overall, 62% of peptides identified in CZE-ESI-MS/MS and 67% in UPLC-ESI-MS/MS were unique. CZE-ESI-MS/MS favored basic and hydrophilic peptides with low molecular masses. Combining the two data sets increased the number of unique peptides by 53%. Our approach identified more than twice as many proteins as the previous record for capillary electrophoresis proteome analysis. CE-ESI-MS/MS is a useful tool for the analysis of proteome samples of intermediate complexity.

Fully Automated Two-dimensional Capillary Electrophoresis for High Sensitivity Protein Analysis

We report a system for automated protein analysis. In the system, proteins are labeled with the fluorogenic reagent 3-(2-furoyl)quinoline-2-carboxaldehyde, which reacts with lysine residues and creates a highly fluorescent product. These labeled proteins are analyzed by submicellar capillary electrophoresis at pH 7.5 to perform a first dimension separation. Once the first components migrate from the capillary, a fraction is transferred to a second dimension capillary, where electrophoresis is performed at pH 11.1 to further separate the proteins. Laser-induced fluorescence is used as an ultrasensitive detector of the separated proteins. Successive fractions are transferred from the first dimension capillary to the second dimension capillary for further separation to generate, in serial fashion, a two-dimensional electropherogram. The transfer of fractions is computer-controlled; there is no operator intervention once the sample has been injected. Zeptomoles of labeled proteins are detected, providing exquisite sensitivity.

Ultrasensitive and Fast Bottom-up Analysis of Femtogram Amounts of Complex Proteome Digests**

Femtogram proteomics: An ultrasensitive capillary zone electrophoresis-mass spectrometry system that is based on an improved nanospray interface has been developed. This system is used for the analysis of picogram to femtogram amounts of E. coli digests; for example, over 100 proteins were identified from 16 pg digests by tandem mass spectrometry. AMTs=accurate mass and time tags.

Correlating Cell Cycle With Metabolism in Single Cells: Combination of Image and Metabolic Cytometry

BACKGROUND We coin two terms: First, chemical cytometry describes the use of high-sensitivity chemical analysis techniques to study single cells. Second, metabolic cytometry is a form of chemical cytometry that monitors a cascade of biosynthetic and biodegradation products generated in a single cell. In this paper, we describe the combination of metabolic cytometry with image cytometry to correlate oligosaccharide metabolic activity with cell cycle. We use this technique to measure DNA ploidy, the uptake of a fluorescent disaccharide, and the amount of metabolic products in a single cell. METHODS A colon adenocarcinoma cell line (HT29) was incubated with a fluorescent disaccharide, which was taken up by the cells and converted into a series of biosynthetic and biodegradation products. The cells were also treated with YOYO-3 and Hoechst 33342. The YOYO-3 signal was used as a live-dead assay, while the Hoechst 33342 signal was used to estimate the ploidy of live cells by fluorescence image cytometry. After ploidy analysis, a cell was injected into a fused-silica capillary, where the cell was lysed. Fluorescent metabolic products were then separated by capillary electrophoresis and detected by laser-induced fluorescence. RESULTS Substrate uptake measured with metabolic cytometry gave rise to results similar to those measured by use of laser scanning confocal microscopy. The DNA ploidy histogram obtained with our simple image cytometry technique was similar to that obtained using flow cytometry. The cells in the G(1) phase did not show any biosynthetic activity in respect to the substrate. Several groups of cells with unique biosynthetic patterns were distinguished within G(2)/M cells. CONCLUSIONS This is the first report that combined metabolic and image cytometry to correlate formation of metabolic products with cell cycle. A complete enzymatic cascade is monitored on a cell-by-cell basis and correlated with cell cycle.