news NEWS FROM THE 10TH ANNUAL FREDERICK CONFERENCE ON CAPILLARY ELECTROPHORESIS Britt Erickson reports from Hood College, Frederick, MD.
Beyond proteomics First there was genomics, then proteomics, now “physiomics”—the study of enzyme activity within single cells. Nancy L. Allbritton and her colleagues at the University of California–Irvine are among a handful of researchers investigating what happens in the millisecond-to-second time scale following the activation of key regulatory proteins. They have developed a method for simultaneously measuring the activation of multiple enzymes in response to pharmacologic and physiologic stimuli in single cells. Because enzymatic activity changes outside the intracellular environment, one of the key challenges was developing a method that could determine activities and functional relationships of multiple elements within intact networks, says Allbritton. To accomplish this, the researchers combined a rapid cytoplasmic sampling technique, which completely lyses a single cell in < 33 ms, with CE. The sampling technique uses a laser–micropipet combination that takes advantage of a shock wave produced by a highly focused, pulsed laser microbeam (Anal. Chem. 1998, 70, 4570–4577). Upon lysis, the cellular contents are loaded into a standard capillary by a combination of gravity flow and/or electrophoresis. Protein kinase C (PKC) activation was measured by microinjecting a fluorescently labeled PKC–substrate peptide into a cell. After varying incubation times, the cytoplasm (pL volume) was loaded into a capillary using the rapid cytoplasmic sampling technique. The nonphosphorylated and phosphorylated substrate peptides were separated by CE and quantified using laser-induced fluorescence (LIF). Detection limits were < 10–19 mol of peptide. The ratios of phosphorylated to nonphosphorylated substrate peptides
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were used to measure the activation of PKC. A mixture of substrate peptides with different kinase specificities, including peptides for PKC, calcium-calmodulin-activated kinase II, and cdc2 protein kinase, was injected into the same cell and separated Schematic of system used for cell lysis, CE, and LIF. by CE. All three peptides were detected in the electropherogram. According to Allbritton, the method should be applicable to single cell other single cell measurements, such as measurements for a wide range of Ca2+ imaging. The method, she says, has great potential for unraveling several enzymes, including phosphatases, procomplex signaling networks that conteases, and nucleases. In addition, it trol cellular responses. can be performed concurrently with
Fluorescence detection on plastic microchips In their quest for inexpensive, disposable microchips, researchers have turned to a variety of synthetic polymers, including co-polyesters, acrylics, and polystyrene, as alternatives to silicon or glass. The problem with using these thermoplastic chips for microseparations is that the plastics themselves fluoresce, resulting in large background signals when fluorescence detection is used. To reduce background fluorescence, confocal optical systems with high numerical aperture (NA) optics are needed. These systems, however, work best with coherent light sources, such as lasers, which are quite expensive. In an effort to keep costs down, Shau-Chun Wang and Michael D. Morris of the University of Michigan have developed a fluorescence detection strategy for electrophoretic separations on co-polyester microchips that
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works well with low-NA optics and is effective with an inexpensive blue (485-nm) LED light source. Referred to as analyte velocity modulation with lock-in synchronous demodulation, the method easily separates the analyte signal from the background signal, resulting in an order-of-magnitude improvement in detection limits compared with DC detection. In analyte velocity modulation, the driving DC voltage is modulated at a low frequency (typically 7–20 Hz), such that the velocity of ions migrating through the channel and the analyte signals are modulated at the same frequency. The fluorescence from the analyte and the microchip is collected through a 10x or 20x microscope objective and passed through a long pass filter and aperture to a photomultiplier tube. Because the microchip
