TY - JOUR
T1 - Native mass spectrometry and gas-phase fragmentation provide rapid and in-depth topological characterization of a PROTAC ternary complex
AU - Song, Jong Hee
AU - Wagner, Nicole D.
AU - Yan, Jing
AU - Li, Jing
AU - Huang, Richard Y.C.
AU - Balog, Aaron J.
AU - Newitt, John A.
AU - Chen, Guodong
AU - Gross, Michael L.
N1 - Funding Information:
The authors thank Susan E. Kiefer, Frank Marsilio, Weifang Shan, Drs. Mark Witmer, Louis Lombardo, and Olafur Gudmundsson of Bristol Myers Squibb Company for their support of this project and the members of M.L.G. lab for assistance. This work was supported by a research collaboration with Bristol Myers Squibb Company and by the National Institutes of Health (P41GM103422 and R24GM136766). J.H.S. and J.Y. designed the experiments and acquired results. J.L. and G.C. provided ternary complex materials and assisted with experimental design. J.H.S. and N.D.W. analyzed and interpreted the results with input from all authors. J.H.S. N.D.W. and M.L.G. wrote the manuscript with input from all authors. M.L.G. supervised the research. Michael L. Gross is an unpaid member of the scientific advisory boards of Protein Metrics Inc. and Gen Next Technologies, two companies pursuing ideas in structural mass spectrometry.
Funding Information:
The authors thank Susan E. Kiefer, Frank Marsilio, Weifang Shan, Drs. Mark Witmer, Louis Lombardo, and Olafur Gudmundsson of Bristol Myers Squibb Company for their support of this project and the members of M.L.G. lab for assistance. This work was supported by a research collaboration with Bristol Myers Squibb Company and by the National Institutes of Health ( P41GM103422 and R24GM136766 ).
Publisher Copyright:
© 2021 Elsevier Ltd
PY - 2021/10/21
Y1 - 2021/10/21
N2 - Proteolysis-targeting chimeras (PROTACs) represent a new direction in small-molecule therapeutics whereby a heterobifunctional linker to a protein of interest (POI) induces its ubiquitination-based proteolysis by recruiting an E3 ligase. Here, we show that charge reduction, native mass spectrometry, and gas-phase activation methods combine for an in-depth analysis of a PROTAC-linked ternary complex. Electron capture dissociation (ECD) of the intact POI-PROTAC-VCB complex (a trimeric subunit of an E3 ubiquitin ligase) promotes POI dissociation. Collision-induced dissociation (CID) causes elimination of the nonperipheral PROTAC, producing an intact VCB-POI complex not seen in solution but consistent with PROTAC-induced protein-protein interactions. In addition, we used ion mobility spectrometry (IMS) and collisional activation to identify the source of this unexpected dissociation. Together, the evidence shows that this integrated approach can be used to screen for ternary complex formation and PROTAC-protein contacts and may report on PROTAC-induced protein-protein interactions, a characteristic correlated with PROTAC selectivity and efficacy.
AB - Proteolysis-targeting chimeras (PROTACs) represent a new direction in small-molecule therapeutics whereby a heterobifunctional linker to a protein of interest (POI) induces its ubiquitination-based proteolysis by recruiting an E3 ligase. Here, we show that charge reduction, native mass spectrometry, and gas-phase activation methods combine for an in-depth analysis of a PROTAC-linked ternary complex. Electron capture dissociation (ECD) of the intact POI-PROTAC-VCB complex (a trimeric subunit of an E3 ubiquitin ligase) promotes POI dissociation. Collision-induced dissociation (CID) causes elimination of the nonperipheral PROTAC, producing an intact VCB-POI complex not seen in solution but consistent with PROTAC-induced protein-protein interactions. In addition, we used ion mobility spectrometry (IMS) and collisional activation to identify the source of this unexpected dissociation. Together, the evidence shows that this integrated approach can be used to screen for ternary complex formation and PROTAC-protein contacts and may report on PROTAC-induced protein-protein interactions, a characteristic correlated with PROTAC selectivity and efficacy.
KW - CID (collision-induced dissociation)
KW - CIU (collision-induced unfolding)
KW - E3 ligase
KW - ECD (electron capture dissociation)
KW - IMS (ion mobility spectrometry)
KW - MZ1
KW - PROTACs (proteolysis-targeting chimeras)
KW - charge-reducing agent
KW - native mass spectrometry
KW - ternary complex
UR - http://www.scopus.com/inward/record.url?scp=85117213251&partnerID=8YFLogxK
U2 - 10.1016/j.chembiol.2021.05.005
DO - 10.1016/j.chembiol.2021.05.005
M3 - Article
C2 - 34081921
AN - SCOPUS:85117213251
SN - 2451-9456
VL - 28
SP - 1528-1538.e4
JO - Cell Chemical Biology
JF - Cell Chemical Biology
IS - 10
ER -