Microelectrophoretic Techniques for Cellular Kinase

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method by Jorgenson and Lukacs in the 1980s.78 CE uses a small internal diameter, fused silica capillary filled with an electrolytic buffer. Once the capillary is loaded with sample, high voltage is applied across the capillary ends and the components of the sample are separated within the capillary based on their different mobilities in an electric field.

Electroosmotic flow (EOF) arises at the charged capillary wall and works as a mobile “pump” to sweep all species in the capillary toward one terminal. Velocity of migration is

determined by both the individual sample component’s electrophoretic migration and the electroosmotic mobility of the buffer (Figure 1.2).

CE is a well-established technology for protein and peptide analysis. This technique has been successfully applied to protein and peptide separations in several fields, such as the separation of proteolytic fragments and other metabolic products. Combined with mass spectrometry, CE becomes a powerful analytical tool for proteomic studies.79-81 More recently, this technique has been used to analyze the contents of single cells or small cell sample size in the field known as chemical cytometry. Electrophoretic separation combined with laser-induced fluorescence detection (LIF) is one of the most sensitive methods used for protein investigation, enabling numerous biochemical studies in single cells.82-86 For

example, by analyzing a fluorescent protein fused to a caspase substrate expressed in cells, CE-LIF elucidates caspase activation in apoptosis at the single-cell level. Fluorescent peptides have also been used as reporters to investigate enzymatic activities in vitro.87-89 In these studies, CE has the capability to separate phosphorylated and non-phosphorylated peptide reporters based on the differences in electrophoretic mobilities. The rate of phosphorylation can be calculated and used to assess the activity of the kinase. This is calculated by determining the ratio of the peak areas corresponding to phosphorylated and

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non-phosphorylated species in an electropherogram. In addition, CE was also applied to successfully separate the degradation products of peptides,81 making CE a powerful tool for monitoring peptide degradation.90

The Allbritton group has pioneered the development of single-cell assays for chemical cytometry and studied new reagents and instrumentation. To support research on signal transduction, particularly kinase assays,90-97 a system was developed for single-cell assays. This platform integrates an inverted microscope, a pulsed laser, an LIF detection system, and a CE setup to provide several advantages for single cell assays, such as fast cell lysis, high sensitivity, the potential for multiplexed measurements of several kinases, and monitoring of cells in real-time.

Chemical cytometry experiments are performed on this instrument as follows: First, cells are loaded with fluorescently labeled substrates, which act as reporters for the activity of the target enzymes within the cells. The substrates can be fluorescent peptides or lipids. For protein kinase assays, the substrates contain a serine, threonine or tyrosine residue which can be recognized and phosphorylated by target kinases.96 After cells were loaded with substrates and attached to the sample dish, a nono-second pulsed laser was fired at a position close to the cell. The plasma generated by the laser pulse lysed the cell within tens of micro seconds. Immediately after lysis, the cellular contents were electrokinetically injected into the capillary and separated by CE. The reporters are typically labeled with carboxy

fluorescein, detected using LIF, and identified by their characteristic migration times. The ratio of the substrate and product peak areas are calculated and used to measure the targeted enzymes’ activity. The ability to load more than one reporter into a cell at the same time allows activity measurement of multiple enzymes simultaneously. Three kinases have been

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assayed at the same time in single cells so far and substantially greater multiplexing is

possible due to the very high separation efficiency of micro-electrophoretic methods.90,92-94,96- 98

1.4.2 Cytosolic Degradation as a Limitation of Peptide Reporters for Cell Assays Although the peptide reporters have been shown effective, a major pitfall is the degradation by intracellular proteases. This degradation drastically shortens the lifetime of the peptides inside the cell, decreasing their utility as reporters. For example, as the peptide reporter degrades, the signals for the phosphorylated and unmodified forms become spread out over multiple peaks, making them harder to detect.

Intracellular proteolysis is a natural process in the cell which controls the turnover of proteins and the amount of peptides (antigens) present. This degradation process typically involves the ubiquitin-proteasome system, as well as several aminopeptidases and

endopeptidases. In eukaryotic cells, a protein is first degraded by the 26S proteasome into oligopeptides that are either the correct size for antigenic peptides or extended on their N- termini. Aminopeptidases in the cytosol or ER trim the N-extended precursors into antigenic peptides of the correct length. Proteosome products and other oligopeptides undergo

degradation by endo- and exopeptidases. With the effect of the combination of the above process, a peptide reporter’s stability within the cell is limited.99-103

Key cytoplasmic peptidases include tripeptidylpeptidase II (TPPII), thimet

oligopeptidase (TOP), prolyl oligopeptidase (POP), and leucine aminopeptidase (LAP). It has been found that a common feature of these peptidase structures is a deep cleft or a tunnel in which the catalytic site is buried. Thus, for a peptide to be degraded, it must be threaded into a cleft or through a tunnel. In endopeptidases, such as TOP, the amino terminus of the

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substrate peptide is buried deep within the cleft.102,104,105 This dissertation focuses on utilizing this aspect of peptidase structure to slow peptide degradation in cells.

1.5 Research Goals and Scope of the Dissertation