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Combining Absolute Quantitative Cross-Linking Mass Spectrometry and Molecular Modeling for Probing PROTAC-Mediated Ternary Complex Structures

Award Number: R21GM148874
ORGANIZATION: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES
OPDIV: NIH
AWARD CLASS: DISCRETIONARY
AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)
PERIOD OF PERFORMANCE START DATE: 03/01/2023
PERIOD OF PERFORMANCE END DATE: 02/28/2026

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Combining Absolute Quantitative Cross-Linking Mass Spectrometry and Molecular Modeling for Probing PROTAC-Mediated Ternary Complex Structures - 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.


Issue Date FY	Funding FY	Legal Entity Name	Legal Entity Address	Legal Entity City	Legal Entity State	Legal Entity Zip Code	Legal Entity COUNTY	Legal Entity COUNTRY	Assistance Listing	Award Code	Budget Year	Action Date	Action Type	Action Amount

Issue Date FY: 2025 ( Subtotal = $0 )
2025	2024	NEW JERSEY INSTITUTE OF TECHNOLOGY	323 DR MARTIN LUTHER KING JR BLVD	NEWARK	NJ	07102	ESSEX	USA	Biomedical Research and Research Training	000	2	1/31/2025	NON-COMPETING CONTINUATION	$0
														Subtotal = $0

Issue Date FY: 2024 ( Subtotal = $189,312 )
2024	2024	NEW JERSEY INSTITUTE OF TECHNOLOGY	323 DR MARTIN LUTHER KING JR BLVD	NEWARK	NJ	07102	ESSEX	USA	Biomedical Research and Research Training	000	2	2/19/2024	NON-COMPETING CONTINUATION	$170,380
2024	2024	NEW JERSEY INSTITUTE OF TECHNOLOGY	323 DR MARTIN LUTHER KING JR BLVD	NEWARK	NJ	07102	ESSEX	USA	Biomedical Research and Research Training	001	2	6/18/2024	NON-COMPETING CONTINUATION	$18,932
														Subtotal = $189,312

Issue Date FY: 2023 ( Subtotal = $239,990 )
2023	2023	NEW JERSEY INSTITUTE OF TECHNOLOGY	323 DR MARTIN LUTHER KING JR BLVD	NEWARK	NJ	07102	ESSEX	USA	Biomedical Research and Research Training	000	1	2/22/2023	NEW	$239,990
														Subtotal = $239,990

Grand Total All Awards = $429,302

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Combining Absolute Quantitative Cross-Linking Mass Spectrometry and Molecular Modeling for Probing PROTAC-Mediated Ternary Complex Structures

Award Number: R21GM148874

ORGANIZATION: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES

OPDIV: NIH

AWARD CLASS: DISCRETIONARY

AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)

PERIOD OF PERFORMANCE START DATE: 03/01/2023

PERIOD OF PERFORMANCE END DATE: 02/28/2026

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