A Modular Cross-Linker Platform for Proteome-Scale Mapping of Dynamic Protein-Protein Interactions in Cells

About This Grant

PROJECT SUMMARY Protein–protein interactions (PPIs) govern nearly every facet of cellular function and represent a key axis for therapeutic targeting. Yet, the vast majority of the ~650,000 predicted PPIs in the human proteome remain uncharacterized, largely due to limitations in technologies that can map interactions in live cells. Existing approaches rely on genetic tagging or in vitro systems and lack the resolution to detect transient, low-abundance, or state-specific interactions—especially in pharmacologically or disease-relevant contexts. This proposal aims to overcome these challenges by developing a next-generation crosslinking mass spectrometry (XL-MS) platform that enables quantitative, proteome-wide interactome mapping in live cells. Current XL-MS methods are limited by narrow residue reactivity, short and inflexible spacer geometries, inefficient enrichment strategies, and poor quantitative compatibility. Here, we will build a modular crosslinker platform that enables: (i) orthogonal residue reactivity and tunable spacer designs to access a broader spectrum of interaction interfaces; (ii) isotope-encoded handles for robust MS1-based quantitation; and (iii) cleavable enrichment handles that allow gentle, MS- compatible peptide recovery. In Aim 1, we will synthesize and benchmark a chemically diverse crosslinker library, systematically evaluating how spacer length, geometry, and residue reactivity affect proteome-wide PPI coverage in cells. In Aim 2, we will develop isotopically encoded variants and optimized workflows for quantitative PPI comparison across biological states, applying the platform to two high-impact systems: unbiased mapping of small molecule–modulated PPIs (stabilizers and disruptors) and dynamic PPI remodeling during human T cell activation. Together, these studies will establish a versatile XL-MS platform and reagents that addresses key limitations of current technologies, enabling quantitative, proteome-scale PPI discovery in native cellular contexts with unprecedented sensitivity, coverage, and versatility.