Administrative Supplement to Characterizing proteasome-substrate interactions by mass spectrometry proteomics

项目总结

PROJECT SUMMARY We are proposing to purchase a Thermo FAIMS Pro Orbitrap Ion Source that will attach to the front of a Lumos Orbitrap and be used in our ubiquitin-proteasome cross-linking experiments. Our research project involves probing interactions between polyubiquitin chains, model proteasome substrates and the yeast proteasome. Cross-linking mass spectrometry is being applied to discover novel ubiquitin receptors and to better understand how tagged substrates can impact proteasome function. It is crucial that cross-links are identified with high confidence because of the complexity of the biochemical assays that build on these results. Since there is limited consensus in this field as to what constitutes an unambiguous cross-link identification, we are exerting considerable effort to improve cross-linking observation and interpretation methodology. By combining electron transfer dissociation, collision induced dissociation, and high energy collision induced dissociation of precursor ions we have recently reported how the credibility of cross-link identifications can be enhanced. Crucial to the success of this approach is to have intense precursor ion signals. The Thermo FAIMS Pro Orbitrap Ion Source is designed to increase the transmission of highly-charged ions such as those associated with cross- linked peptides and to discriminate against background species such as individual peptides. In fact, it has already demonstrated success in cross-linking mass spectrometry experiments. We will use this device to discover and confirm cross-links involving polyubiquitin and the yeast proteasome and by doing so will achieve both analytical and biological advances. Progress in our ability to recognize and conclusively identify cross-linked peptides has transformative potential for elucidating some of the most important and challenging issues in protein science. Specifically, our cross-linking proteomics experiments will provide candidates for novel substrate receptors on the proteasome and will identify the sizes and linkage patterns in polyubiquitin that are most effective for recognition by the proteasome. In summary, this project should result in significant advances in cross-linking methodology and in our understanding of how the proteasome recognizes and digests substrates.