iProXProteomicsLihua ZhangHomo Sapienshttp://www.iprox.org/page/project.html?id=IPX0005492000lihuazhang@dicp.ac.cnDalian Institute of Chemical Physics, Chinese Academy of ScienceiProXComprehensive interactome analysis of targeted proteins is important to understand how proteins work together in regulating functions. Commonly, affinity purification followed by mass spectrometry (AP-MS) has been recognized as the most often used technique for studying protein-protein interactions (PPIs). However, some proteins with weak interactions, which are responsible for key roles in regulation, are easily broken during cell lysis and purification through an AP approach. Herein, we have developed an approach termed in vivo cross-linking-based affinity purification and mass spectrometry (ICAP-MS). By this method, in vivo cross-linking was introduced to covalently fix intracellular PPIs in their functional states to assure all PPIs could be integrally maintained during cell disruption. In addition, the chemically cleavable crosslinkers which were employed enabled unbinding of PPIs for in-depth identification of components within the interactome and biological analysis, while allowing binding of PPIs for cross-linking-mass spectrometry (CXMS)-based direct interaction determination. Multi-level information on targeted PPIs network can be obtained by ICAP-MS, including composition of interacting proteins, as well as direct interacting partners and binding sites. As a proof of concept, the interactome of MAPK3 from 293A cells was profiled with 6.15-fold improvement in identification than by conventional AP-MS. Meanwhile, 184 cross-link site pairs of these PPIs were experimentally identified by CXMS. Furthermore, ICAP-MS was applied in the temporal profiling of MAPK3 interactions under activation by cAMP-mediated pathway. The regulatory manner of MAPK pathways was presented through the quantitative changes of MAPK3 and its interacting proteins at different time points after activation. Therefore, all reported results demonstrated that the ICAP-MS approach may provide comprehensive information on interactome of targeted protein for functional exploration.In vivo cross-linking-based affinity purification and mass spectrometry for targeting intracellular protein-protein interactions.Zhong Bowen B, An Yuxin Y, Gao Hang H, Zhao Lili L, Li Xiao X, Liang Zhen Z, Zhang Yukui Y, Zhao Qun Q, Zhang Lihua LfalseIn-vivo crosslinking based affinity purification-mass spectrometry for targeting intracellular protein-protein interactionsComprehensive interactome analysis of targeted proteins is of great significance to understand how pro-teins work together for functional regulation. Until now, affinity purification strategy with mass spec-trometry (AP-MS) has been recognized as the most widely used technique for studying protein-protein interactions (PPIs). However, some proteins with weak interactions, responsible for key roles in pathway regulation, are easily broken during cell lysis by AP approach. To advance the identification depth of protein interactome, herein we developed an approach named in vivo crosslinking based affinity purifi-cation-mass spectrometry (ICAP). By this method, in vivo crosslinking was introduced for covalently fixing the intracellular PPIs at their functional states to assure all the PPIs could be integrally maintained during cell disruption. Contributed by this advantage, the transient/weak PPIs missed in the AP-MS method could be captured in the consecutive biotin-streptavidin purification. With ICAP approach, the interactome of ERK1 from 293A cells was profiled with 6.15-fold improvement in identification number than those by conventional AP-MS. Contributed by the superior performance, ICAP was further extend-ed to the temporal profiling of ERK1 interacting proteins under activation by cAMP-mediated pathway. The reasonable regulatory manner of MAPK pathways was explored by depicting the quantitative changes of ERK1 and its interacting proteins at different time points after activation. Furthermore, owing to chemical cleavable crosslinkers-DSEP, we can choose to perform CXMS analysis for identification of specific protein-protein interactions and sites within interacting proteins in the network. Therefore, all these results demonstrated that our approach can provide comprehensive information on interactome of target protein for molecular biologists in a variety of diverse applications.Thu Dec 01 00:00:00 GMT 2022PXD038473960637230567