3 years ago

Quantitative and Comparative Profiling of Protease Substrates through a Genetically Encoded Multifunctional Photocrosslinker

Quantitative and Comparative Profiling of Protease Substrates through a Genetically Encoded Multifunctional Photocrosslinker
Peng R. Chen, Fan Yang, Xiao Xie, Dan He, Haiping Song, Yun Ge, Heng Zhang, Haomiao Su
A genetically encoded, multifunctional photocrosslinker was developed for quantitative and comparative proteomics. By bearing a bioorthogonal handle and a releasable linker in addition to its photoaffinity warhead, this probe enables the enrichment of transient and low-abundance prey proteins after intracellular photocrosslinking and prey–bait separation, which can be subject to stable isotope dimethyl labeling and mass spectrometry analysis. This quantitative strategy (termed isoCAPP) allowed a comparative proteomic approach to be adopted to identify the proteolytic substrates of an E. coli protease–chaperone dual machinery DegP. Two newly identified substrates were subsequently confirmed by proteolysis experiments. A genetically encoded trifunctional photocrosslinker was developed that bears a bioorthogonal handle and a releasable linker in addition to its photoaffinity warhead. This probe enabled the enrichment of transient and low-abundance prey proteins after intracellular photocrosslinking and prey–bait separation. A quantitative and comparative proteomic strategy using this probe revealed the substrates of a protease.

Publisher URL: http://onlinelibrary.wiley.com/resolve/doi

DOI: 10.1002/anie.201708151

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