20201005 Applications of Top Down Proteomics

The slides for this lecture can be downloaded here: https://drive.google.com/file/d/1l-Ed...

The African Centre for Gene Technologies asked me to lecture on the applications of top-down proteomics as part of capacity development for biotechnology in South Africa. After spending a couple weeks with the rapidly growing literature in this area, I was excited to talk about what I learned!

This lecture is the biological counterpart to my lecture on Informatics for Top-Down Proteomics (   • 20200512 Bioinformatics Top Down Prot...  ). I started with an explanation on why top-down tandem mass spectra are so much more complex than those we typically see in bottom-up "shotgun" experiments, and we examined three different visualizations that frequently appear to illustrate Proteoform Spectrum Matches (PrSMs): isotopic envelopes from MS scans, Roepstorff Fohlman fragment maps, and annotated MS/MS scans. We briefly visited methods for separating proteoforms by size (size exclusion chromatography, ion mobility, and FELFrEE), by pH or charge (isoelectric focusing and capillary zone electrophoresis), and in LC (particularly ion exchange chromatography and hydrophobic / hydrophilic interaction chromatography).

For applications of top-down, I emphasized six different "Use Cases" that represent common biological questions for top-down:
1. Which PTMs co-occur across the proteoform?
2. Which transcript is expressed here?
3. Are all parts of the sequence present in mature form?
4. Do proteoforms differ under various conditions?
5. How pure is this protein preparation?
6. Is this protein part of a larger molecular machine?

Particularly for the last of these questions, I mentioned some combined-methods routes to employ top-down techniques, such as "rare-cutter" proteases or other reagents that produce much longer peptides and integrated "TDBU" techniques that combine information from top-down and bottom-up experiments. As we closed, I explained some of the pioneering work now taking place in "Native MS" that seeks to recognize the protein-protein interactions native to the cellular environment.