Tools for in vivo Proteomics

Applications

This is a tool for enhancing the spatiotemporal resolution of mass spectrometry-based proteomics by tagging cellular proteins in vivo.

Problem Addressed

Mass spectrometry (MS) is a tool essential to elucidating the complex relationships between cellular molecules. However, current methods have only limited spatiotemporal resolution, and suboptimal sample preparation may lead to the introduction of biological artifacts. 

Technology

Professor Alice Ting and colleagues have developed an approach that involves genetically fusing an engineered ascorbate peroxidase (APEX) to a protein of interest to catalyze the addition of a biotin-phenol compound to proteins in its immediate proximity. This reaction occurs quickly (<1 minute), thereby preserving any time-dependent spatial relationships. Biotinylated proteins can be recovered after cell lysis with streptavidin beads, digested, and identified with tandem MS/MS. The uses and advantages of the biotin-phenol compound extend beyond MS, since it will bind to streptavidin or avidin conjugated to a variety of molecules. For example, proteins labeled with biotin-phenol can be tagged with fluorophore-conjugated streptavidin and visualized via microscopy, providing a means to relate results from mass spectrometry with those from imaging. Thus, this in vivo labeling technique is a highly versatile and adaptable method for precisely interrogating cellular protein dynamics.

Advantages

  • Nanometer spatial resolution
  • 1 minute temporal resolution
  • Avoid artifacts arising from biochemical fractionation, including loss of protein, and introduction of contaminants
  • Genetic tag is small, sterically compatible with most proteins, and can be expressed in all cellular compartments and all cell types (bacteria, yeast, mammalian cells, neurons, etc.)

Related Technologies

APEX for EM is a related technology.