Improving 2D Gel Proteomics With The Structured Illumination Gel Imager

This thesis is composed of three separate projects: 1. Proteomics is the study of complex protein mixtures found in a cell, organ, or entire organism. The vast concentration range of these samples, estimated at approximately 150,000-fold for simple unicellular eukaryotes is beyond current detection methods. We present a technology called Structured Illumination (SI) Gel Imager that employs an LCD projector to selectively illuminate fluorescently labeled proteins separated into individual protein spots on 2-dimensional electrophoresis (2DE) gels. SI Gel Imager images have a dynamic range of approximately 1,000,000-fold, making it a valuable tool for proteomic detection. 2. 2DE gels possess the ability to separate proteins with extremely high resolution of molecular-weight and isoelectric-point. However, they suffer from variable sample loss incurred during protein reduction and alkylation steps required for subsequent sequencing by mass-spectrometry, up to about 30% of the starting sample. We present a protein equilibration method utilizing agarose stacking gels to reduce experiment variability and sample loss. 3. 2DE-based proteomics is a time-consuming process, requiring up to 3 days, and suffers from low reproducibility. To provide undergraduates to the experience of conducting proteomics research, we developed the Proteomics Platoon approach, where a group of undergraduate students work in two-person teams to perform proteomic experiments using a wide variety of biological samples. The close-knit nature of the Platoon further fosters collaboration, communication and mentorship while completing complex scientific projects. The Platoon approach serves as a model for involving undergraduates in complex research projects.