Development of a Novel Assay of Protein Tyrosine Phosphatase Activity in Single Cells Using Capillary Electrophoresis

RYAN MATTHEW PHILLIPS: Development of a Novel Assay of Protein Tyrosine Phosphatase Activity in Single Cells Using Capillary Electrophoresis (Under the direction of The inhalation of diesel exhaust particles has been linked to human diseases In vitro studies have implicated the inhibition of protein tyrosine phosphatases (PTPs) by diesel exhaust components as a contributor to the inflammatory processes underlying these conditions. A more complete mechanistic understanding of this phenomenon could be achieved by observing the effects of diesel exhaust particle inhalation on the airways of exposed living human subjects. While airway specimens can be obtained safely from these subjects by bronchial brushing, sample analysis is complicated by low total cell numbers, poor viability, and contamination with inflammatory cells and mucus. We present a novel approach to the analysis of these challenging samples at the single-cell level that provides a direct measure of PTP activity without sacrificing information about intercellular heterogeneity commonly lost by analysis of bulk lysates. A fluorescent phosphopeptide substrate of PTPs was synthesized and an analytical separation was developed to resolve and quantify phosphorylated and nonphosphorylated peptide, as well as potential degradation products, using capillary electrophoresis with laser-induced fluorescence detection. Peptide dephosphorylation was then used as a measure of PTP activity in a variety of model systems including recombinant PTPN1 and PTPN2, A431 cell lysates, and single A431 cells. The reporter was also used to characterize the inhibition iii of PTP activity in these systems by three toxic components of diesel exhaust particles: pervanadate, 1,2-naphthoquinone, and Zn 2+. The PTP reporter was then applied to common model systems of airway biology. PTP activity, as well as inhibition by the toxins listed above, was quantified in immortalized BEAS-2B bronchial epithelial cells as well as cultured primary human airway epithelial cells. PTP activity was then measured in single cells obtained directly from a living human subject via bronchial brushing. The ability to analyze these small, heterogeneous samples of primary cells at the single-cell level demonstrates the power of this approach as new tool for the field of airway biology as well as for the broader study of PTP signaling.