We are developing a modular microfluidic pipeline for the loss-less (no blotting steps) sample preparation of nanoliter volumes for negative stain and cryo-EM. In particular, to extract the entire cytosol of a single cell, deposit it in a loss-less manner on an EM grid, and visualize it by transmission electron microscopy (TEM) at sufficient resolution to recognize proteins and protein complexes by computer image analysis.
1. To quantitatively study the aggregation of proteins that occurs in diseases such as Parkinson’s and Alzheimer’s.
2. To carry out single cell proteomics.
3. To obtain quantitative data on the relative occurrences of the proteins and protein complexes in the cell, even when these are present at low copy number.
4. To purify targeted proteins from the cell lysate by affinity-isolation, and investigate them by interaction-labeling to unravel reaction pathways.
Importance: The loss-less, visual inspection of the cell's entire cytosol and the ability of TEM to detect very few single molecules of a given component, provide the extreme sensitivity important for single-cell analysis.
The sensitivity and the structural information delivered by the method are key to the study of neurodegeneration, where disease initiation and progression at the cellular level is little understood.
Why are in vitro studies required: To fully understand the aggregation process it is essential to quantitatively describe the aggregation process and obtain the high-resolution structures of the various filamentous aggregates formed. The former is can only be done ‘in vitro’ ‘.
The Visual Proteomics Pipeline being developed is geared to automation, as many single cells must be examined to obtain a true picture of the population and its heterogeneity. For example, protein expression levels vary from cell to cell, cells can respond differently to a given stimulus or, as in neurodegenerative diseases, only some cells in a population might be ‘diseased’. Automation will also facilitate in vitro studies where a large range of conditions have to be screened.