The main focus for our Top Down Proteomics subgroup is to push the limits for whole proteome analysis of mammalian cells, striving for a future in which Top Down analysis rivals that of Bottom Up in the number of protein identifications per run. Recently we have seen progress towards this very goal with the introduction of a separation platform specifically designed to minimize the most common problem in Top Down Proteomics, intact protein separations. This platform effectively reduces sample complexity and separates proteins depending on size, resulting in an opportunity for the researcher to select the optimal analysis method for the sample.
Unlike conventional bottom-up proteomics, in which proteins are digested into peptides and then identified by liquid chromatography and tandem mass spectrometry, top-down proteomics involves the identification of proteins directly from fragmentation of an intact protein, offering richer data around a protein and its molecular composition.
The tectonic collision of biology with separation science, MS, and informatics occurred over the past 15 years and was driven by contributions from more than 100 laboratories. Like budding yeast, MS is sprouting emergent approaches for the direct profiling and MS/MS analysis of heterogeneous proteins in ever more complex mixtures. Such approaches promise to determine molecular indicators of complex diseases and deepen our understanding of dynamic regulatory mechanisms in cell biology.
Toward this effort develops the cell based top-down MS approach to the Human Proteome Project (CB-HPP). By focusing on protein primary structure as expressed in specific cell types, this concept for a cell-based version of the Human Proteome Project is crafted in a manner analogous to the Human Genome Project while recognizing that cells provide a primary context in which to define a proteome. Several activities flow from this articulation of the HPP, which enables the definition of clear milestones and deliverables. The CB-HPP highlights major gaps in our knowledge regarding cell heterogeneity and protein isoforms, and calls for development of technology that is capable of defining all human cell types and their proteomes. The main activities will involve mapping and sorting cell types combined with the application of beyond the state-of-the art in protein mass spectrometry. Learn more about the CB-HPP here. Learn more about top-down proteomics here.
Workflow for High-Throughput Top-Down Proteomics (read more here)
In the Top Down Proteomics platform, a whole cell lysate or nuclear extract go through three different separation steps prior to analysis by mass spectrometry. The first separation (solution isoelectric focusing) separates the proteins in the sample based on their pI. Each of the resulting fractions is then separated by GELFrEE (a size based separation). The next set of fractions are cleaned to remove SDS and separated by reversed phase liquid chromotography prior to MS analysis. Protein identification and characterization are completed with ProSightPC.
Powerful Separation Techniques for Sample Preparation (read more here)
Visualization of 4 mg yeast protein separation by isoelectric focusing (red panel) and subsequent GELFrEE separation of each sIEF fraction (brown panel) by SDS-PAGE. Fractions were eluted off the GELFrEE apparatus and a portion of each fraction was loaded onto gel for visualization.