Supplement for Optically Gated Discovery of Protein-Biomolecule Interactions project.

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The overall goal of research in the Geri lab is to map protein interactomes using discovery technologies that provide orders of magnitude improvements in spatiotemporal resolution over the current state-of-the-art. The motivation for this work is that advancing the resolution of protein interactome discovery technology beyond key milestones, such as single cell and single protein thresholds, will have a field-wide impact analogous to similar advances in transcriptomics and microscopy. Our unified approach, funded through a R35 MIRA award, combines photocatalytic proximity labeling, in which light-powered catalysts attached to an affinity handle drive the crosslinking of synthetic affinity probes with nearby proteins, with patterned light and interaction-gated activation to simultaneously enforce multiple dimensions of specificity. The first year of work in the lab has focused on two of our proposed aims: (1) high throughput discovery of endogenous peptide binding targets, and (2) single-cell resolved spatial interactomics. Endogenous peptides represent a major fraction of proteinaceous matter in human cells, but the mechanisms powering their biological functions remain virtually unexplored. One thrust of our work merges photocatalytic proximity labeling with rapid ion-mobility proteomics to establish their receptors, mapping the interface between the peptidome and the proteome. Key to this effort is the efficient synthesis of catalyst-peptide conjugates. We demonstrated the preparation and isolation of up to ten conjugates in 3 hours using mass directed prep HPLC, which can support up to 800 target ID experiments per year, and demonstrated peptide target ID through photocatalytic labeling and proteomics. Our work to develop spatial interactomics technology has also made substantial progress. We have built bespoke hardware, software, and reagents and used these to demonstrate machine vision-guided patterned irradiation and photocatalytic protein labeling of CD3+ cells in tonsil at single-cell resolution. These projects, and future work in the scope of the R35 MIRA award, entails significant of routine and high throughput organic synthesis and purification, with many intermediates and final products requiring preparatory mass directed HPLC. In the original R35 MIRA application we proposed to use, and during the first year of award activities we have used, a prep HPLC-MS (Waters AutoPurification System 2000 Series) owned by a neighboring laboratory. However, this group (Tri-I TDI) is moving from Weill Cornell Medicine to Rockefeller University, and this instrument will no longer be available. Due to the unexpected nature of their departure from the institution and the lack of any equivalent instrument in a core facility or PI-led laboratory at Weill Cornell Medicine, we are requesting supplemental funding to purchase a replacement instrument (a new Waters AutoPurification System), which costs less than $250,000. We are requesting funding for the entire cost of the instrument and necessary accessories, show that current grant support of the Geri lab is insufficient to purchase the instrument without an administrative supplement, and that sufficient funding is available to support, maintain, and operate the instrument to be purchased.