2. Academic Validation
  3. A Clickable APEX Probe for Proximity-Dependent Proteomic Profiling in Yeast

A Clickable APEX Probe for Proximity-Dependent Proteomic Profiling in Yeast

  • Cell Chem Biol. 2020 Jul 16;27(7):858-865.e8. doi: 10.1016/j.chembiol.2020.05.006.
Yi Li 1,  Caiping Tian 2,  Keke Liu 2,  Ying Zhou 1,  Jing Yang 3,  Peng Zou 4
Affiliations

Affiliations

  • 1 College of Chemistry and Molecular Engineering, Synthetic and Functional Biomolecules Center, Beijing National Laboratory for Molecular Sciences, Key Laboratory of Bioorganic Chemistry and Molecular Engineering of Ministry of Education, Peking University, Beijing 100871, China.
  • 2 State Key Laboratory of Proteomics, National Center for Protein Sciences, Beijing Proteome Research Center, Beijing Institute of Lifeomics, Beijing 102206, China.
  • 3 State Key Laboratory of Proteomics, National Center for Protein Sciences, Beijing Proteome Research Center, Beijing Institute of Lifeomics, Beijing 102206, China. Electronic address: yangjing54@hotmail.com.
  • 4 College of Chemistry and Molecular Engineering, Synthetic and Functional Biomolecules Center, Beijing National Laboratory for Molecular Sciences, Key Laboratory of Bioorganic Chemistry and Molecular Engineering of Ministry of Education, Peking University, Beijing 100871, China; Peking-Tsinghua Center for Life Sciences, PKU-IDG/McGovern Institute for Brain Research, Peking University, Beijing 100871, China. Electronic address: zoupeng@pku.edu.cn.
PMID: 32470320 DOI: 10.1016/j.chembiol.2020.05.006
Abstract

The engineered ascorbate peroxidase (APEX) is a powerful tool for the proximity-dependent labeling of proteins and RNAs in live cells. Although widely use in mammalian cells, APEX applications in Microorganisms have been hampered by the poor labeling efficiency of its biotin-phenol (BP) substrate. In this study, we sought to address this challenge by designing and screening a panel of alkyne-functionalized substrates. Our best probe, Alk-Ph, substantially improves APEX-labeling efficiency in intact yeast cells, as it is more cell wall-permeant than BP. Through a combination of protein-centric and peptide-centric chemoproteomic experiments, we have identified 165 proteins with a specificity of 94% in the yeast mitochondrial matrix. In addition, we have demonstrated that Alk-Ph is useful for proximity-dependent RNA labeling in yeast, thus expanding the scope of APEX-seq. We envision that this improved APEX-labeling strategy would set the stage for the large-scale mapping of spatial proteome and transcriptome in yeast.

Keywords

APEX2; RNA; bioorthogonal reaction; mass spectrometry; mitochondria; proteomics; proximity labeling; spatial transcriptomics; yeast.

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