| Of the 6,000 genes encoded by the S. cerevisiae genome, roughly 300 different gene products have been identified by two-dimensional gel electrophoresis. Although 5% of the expressed proteins are detected and monitored using conventional analytical technology, the study of the remaining 95% of proteins that are expressed at low levels is of fundamental interest in biology.; In contrast to high abundance proteins, which are studied in both classic and single cell proteomics, low level regulatory proteins are much more difficult to study. On one hand, they generate very weak signals and are difficult to monitor due to the insufficient sensitivity of the most techniques. On the other hand, their signals can be swamped by the signals from high abundance proteins.; Low-level gene expression is important in many biological pathways, since regulatory proteins, including those that regulate the responses of the cell to its environment and coordinate the phase transition of the cell cycle, often exist transiently in the cell. Proteins are rapidly inactivated once they have accomplished their tasks, presumably by ubiquitin-mediated proteolysis, by change of phosphorylation state, or by formation of an inactive complex with a specific inhibitor.; I developed a technology which enabled the detection of both the cell cycle dependent protein expression and posttranslational modification of low abundance regulatory proteins in single cells of the yeast S. cerevisiae . Further improvement in the instrument sensitivity will also allow us to define the difference between "on" and "off" of gene expression and determine whether degradation of a protein results in its complete absence in a cell.; In this technology, green fluorescent protein (GFP) hybrids are created with low-expression-level regulatory genes by direct recombination into genomic DNA. These products, their posttranslational modifications, and their complexes with other proteins can be observed at the single copy level. Detection is achieved by single cell capillary electrophoresis (CE) coupled with ultrasensitive laser induced fluorescence detection (LIF). The GFP fusion protein technique ensures the 1:1 ratio of expressed protein and GFP tag, which is advantageous over other labeling methods in protein identification and quantification. |
