Volume 56, Issue 46 pp. 14521-14525
Communication

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

Dan He

Dan He

Synthetic and Functional Biomolecules Center, Beijing National Laboratory for Molecular Sciences, Department of Chemical Biology, College of Chemistry and Molecular Engineering, Peking University, Beijing, 100871 China

These authors contributed equally.

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Xiao Xie

Xiao Xie

Synthetic and Functional Biomolecules Center, Beijing National Laboratory for Molecular Sciences, Department of Chemical Biology, College of Chemistry and Molecular Engineering, Peking University, Beijing, 100871 China

These authors contributed equally.

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Fan Yang

Fan Yang

Synthetic and Functional Biomolecules Center, Beijing National Laboratory for Molecular Sciences, Department of Chemical Biology, College of Chemistry and Molecular Engineering, Peking University, Beijing, 100871 China

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Heng Zhang

Heng Zhang

Synthetic and Functional Biomolecules Center, Beijing National Laboratory for Molecular Sciences, Department of Chemical Biology, College of Chemistry and Molecular Engineering, Peking University, Beijing, 100871 China

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Haomiao Su

Haomiao Su

College of Chemistry and Molecular Sciences, Wuhan University, Wuhan, 430072 China

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Yun Ge

Yun Ge

Synthetic and Functional Biomolecules Center, Beijing National Laboratory for Molecular Sciences, Department of Chemical Biology, College of Chemistry and Molecular Engineering, Peking University, Beijing, 100871 China

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Haiping Song

Haiping Song

Synthetic and Functional Biomolecules Center, Beijing National Laboratory for Molecular Sciences, Department of Chemical Biology, College of Chemistry and Molecular Engineering, Peking University, Beijing, 100871 China

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Prof. Peng R. Chen

Corresponding Author

Prof. Peng R. Chen

Synthetic and Functional Biomolecules Center, Beijing National Laboratory for Molecular Sciences, Department of Chemical Biology, College of Chemistry and Molecular Engineering, Peking University, Beijing, 100871 China

Peking-Tsinghua Center for Life Sciences, Peking University, Beijing, 100871 China

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First published: 20 September 2017
Citations: 53

Graphical Abstract

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.

Abstract

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.

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