Abstract Cross-linking mass spectrometry (CLMS) is playing an increasingly important role in determining protein 3D structures. By structural elucidation of cross-links (i.e., cross-linked peptides resulting from digestion of cross- linked proteins or protein complexes), CLMS can determine distance constraints between specific functional groups that can be used to guide structural modeling and docking of proteins and protein complexes. Further quantitative analysis of cross-links would allow exploration of dynamic protein conformational changes in solution, a challenging task for traditional structural biology techniques. However, absolute quantitative analysis of cross-links is very challenging, due to lack of standard cross-linked peptides. To tackle this issue, we propose to develop a novel absolute quantitative cross-linking mass spectrometry (aqCLMS) using coulometric mass spectrometry (CMS), a method recently developed in our laboratory for absolute quantitation without using standards. It is based on electrochemical oxidation of cross-links to produce electric current and the measurement of the oxidation yield by MS, therefore eliminating the need of using any standards for absolute quantitation. An aqCLMS method would enable: 1) quantitative analysis and comparison among all cross-links at one particular or different conformational states, greatly increasing information about residue accessibilities and their distance constraints in comparison with traditional CLMS; 2) evaluating absolute quantities of transient protein complexes that form during a particular biological event, which is difficult to measure using existing techniques. We foresee that our aqCLMS method is very informative in probing dynamic protein conformations, which would have high impact in structural biology and drug discovery. In this project, we propose to use this aqCLMS approach to probe and quantify the conformational structures of PROteolysis TArgeting Chimera (PROTAC)-mediated complexes, specifically BRD4-PROTAC-CRBN. A key step in PROTAC is the formation of an induced protein complex where a degrader molecule recruits an E3 ligase to the protein of interest (POI) to facilitate the ubiquitination of the POI, eventually leading to its proteasomal degradation. Thus, rational design of PROTACs will require a structural understanding of target-PROTAC-E3 ternary complexes, which is, however, still very limited. In this project, in combination with advanced molecular dynamics (MD) simulations, aqCLMS would provide deep insights into the dynamic nature of the PROTAC- mediated ternary structure ensemble beyond what is seen in the crystal structures.

Narrative Cross-linking mass spectrometry (CLMS), a powerful technique for determining protein conformational structures, lacks capability for absolute quantitation of cross-links (i.e., cross- linked peptides). This proposal aims to develop a novel absolute quantitative cross-linking mass spectrometry (aqCLMS) based on the combination of CLMS with coulometric mass spectrometry (CMS). We also propose to apply aqCLMS, coupled with advanced molecular dynamics (MD) simulation, to study dynamic conformations of PROTAC-mediated ternary complexes for improved accuracy for their structural determination.