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.
