Technology Research & Development Project 6: Targeted Top Down Proteomics Abstract The National Resource for Translational and Developmental Proteomics (NRTDP) has invested significant resources into a discovery pipeline that efficiently generates interesting candidate proteoforms for further investigation. While these experiments produce valuable insight into cell and disease biology, they are labor intensive and relatively low throughput, thus rendering application of this pipeline to larger sample cohorts significantly challenging. Therefore, we propose the development of a complementary targeted quantitative workflow enabling further interrogation of interesting proteoforms in a high throughput modality with enhanced sensitivity. Automated immunoprecipitation will provide the foundation of this new platform. Here, we will draw on the experience of our collaborators at Thermo Pierce, who have validated thousands of antibodies and implemented automated immunocapture using the KingFisher Flex system. Once immunoprecipitated, samples will be resolved via liquid chromatography, ultimately transitioning to the CESI 8000 capillary electrophoresis system from Sciex for improved sensitivity. For each target, we will develop a proteoform reaction monitoring (PfRM) assay that validates and quantitates all of the proteoforms present at stoichiometric levels. The resulting data will be processed using a new software platform, PFRM VIEWER, with the finalized PfRM assays made freely available to the scientific community through a portal on the Resource website. These combined innovations will enable the Resource to advance further down the pathway to translational proteomics by confirming observations in larger populations while also providing a roadmap for community members to implement these assays within their own respective research initiatives.
| Martin, Rey W; Des Soye, Benjamin J; Kwon, Yong-Chan et al. (2018) Cell-free protein synthesis from genomically recoded bacteria enables multisite incorporation of noncanonical amino acids. Nat Commun 9:1203 |
| Kenney, Grace E; Dassama, Laura M K; Pandelia, Maria-Eirini et al. (2018) The biosynthesis of methanobactin. Science 359:1411-1416 |
| Swaroop, Alok; Oyer, Jon A; Will, Christine M et al. (2018) An activating mutation of the NSD2 histone methyltransferase drives oncogenic reprogramming in acute lymphocytic leukemia. Oncogene : |
| Davis, Roderick G; Park, Hae-Min; Kim, Kyunggon et al. (2018) Top-Down Proteomics Enables Comparative Analysis of Brain Proteoforms Between Mouse Strains. Anal Chem 90:3802-3810 |
| LeDuc, Richard D; Schwämmle, Veit; Shortreed, Michael R et al. (2018) ProForma: A Standard Proteoform Notation. J Proteome Res 17:1321-1325 |
| Aebersold, Ruedi; Agar, Jeffrey N; Amster, I Jonathan et al. (2018) How many human proteoforms are there? Nat Chem Biol 14:206-214 |
| Turcan, Sevin; Makarov, Vladimir; Taranda, Julian et al. (2018) Mutant-IDH1-dependent chromatin state reprogramming, reversibility, and persistence. Nat Genet 50:62-72 |
| Lyon, Yana A; Riggs, Dylan; Fornelli, Luca et al. (2018) The Ups and Downs of Repeated Cleavage and Internal Fragment Production in Top-Down Proteomics. J Am Soc Mass Spectrom 29:150-157 |
| Fornelli, Luca; Toby, Timothy K; Schachner, Luis F et al. (2018) Top-down proteomics: Where we are, where we are going? J Proteomics 175:3-4 |
| Fisher, Oriana S; Kenney, Grace E; Ross, Matthew O et al. (2018) Characterization of a long overlooked copper protein from methane- and ammonia-oxidizing bacteria. Nat Commun 9:4276 |
Showing the most recent 10 out of 53 publications
