New technologies for quantification of protein copy number and protein-protein interactions : from single plant cells to organelles

Absolute quantification of cell-to-cell variability arising from the stochastic nature of gene expression is concealed within bulk measurement approaches. While microfabricated technologies for single-cell biology have revealed and advanced our understanding of this heterogeneity, limitations remain in transforming this progress for plant biology. Therefore, this thesis provides single plant cell proteomic profiling technologies to challenge conventional bulk cell analysis to further our knowledge in biological variability. Section one details the establishment, of the first known, miniaturised technology undertaking absolute protein quantification in single plant cells and organelles. Fabricated as an array of sub-nanoliter assay chambers containing micro-printed antibody spots, permitting multiplexed proteomic pull-down assays subsequent to the lysis of isolated protoplasts or organelles. Preliminary evaluation, employing a bi-molecular assay to assess fluorescent gene expression in protoplasts, presents novel experimental insight towards synergistic detection advantages through volume reduction coupled with single-molecule fluorescence microscopy. To demonstrate the quantification of unlabeled proteins a tri-molecular assay is developed for Rubisco, providing a new single-cell perspective towards Rubisco expression during mesophyll protoplast development. Section two challenges the restrictions of antibody affinity and experimental throughput, by merging techniques to produce novel technologies. Initially, a coverslip unifying pull-down assay within a microwell array was developed to overcome throughput limitations and is evaluated by quantifying p53 expression in mammalian cell lines. While the pull-down assay faithfully reproduces the precision in quantitative measurements concerning epi-fluorescence microscopy, a more relevant tool, a secondary standard for quantitative fluorescence microscopy is created. Through ratiometrically calibrating chloroplast autofluorescence against the absolute fluorescent protein content of three transgenic cell lines. These technologies permitted measurements, over six orders of magnitude for organelle, plant, and mammalian protein copy numbers, examined in Section three. Suggesting that unperturbed protein abundance does not scale with absolute expression noise, consequential to a linear relationship between the measured variance and mean.