Development of chemical proteomics enabling tissue-selective proteomic and interactomic analysis

Multicellular lives, such as plants and animals, are complex because different cells inside the organism must differentiate into cell types with distinct morphologies and functions. This differentiation process relies on the expression of different sets of proteins in various cell types and their subsequent assembly into molecular machinery through protein-protein interactions. However, our current understanding of such protein-protein interactions often lacks cellular contexts because they were usually studied in an in vitro heterozygous system or in cultured cells isolated from the tissue environment. Effective methods to interrogate protein-protein interactions in a tissue-specific manner are largely missing.

To close this technology gap, we developed a novel chemical biology approach to label proteins from specific cells with a bifunctional amino acid probe that carries both an affinity tag for proteome extraction and a photo-crosslinker for capturing protein-protein interaction. This new method, termed Methionine Analog-based Cell-Specific Proteomics and Interactomics (MACSPI), enables the profiling of tissue-specific proteomes and the identification of tissue-specific protein interactors in intact organisms and can be applied to a wide range of biological problems, such as the study of organ development and disease pathogenesis.

We designed and synthesized an unnatural amino acid that is structurally similar to methionine but has two additional modules, an alkyne group that can be used as a handle for protein purification through "click chemistry" and a diazirine group that mediates covalent linkages of interacting proteins upon photoactivation. This bifunctional probe is named "photo-ANA". Next, we engineered a methionyl-tRNA synthetase (MetRS) variant that is capable of attaching photo-ANA to methionyl-tRNA. By controlling the expression of this MetRS mutant in specific tissues, only proteins from the tissue of interest are labeled by the photo-ANA probe and extracted by affinity purification. Moreover, with photo-crosslinking, protein complexes from specific tissues can be captured and isolated. We applied MACSPI in Caenorhabditis elegans, a model multicellular organism, to selectively label, capture, and profile the proteomes of the body wall muscle and the nervous system, which led to identifying new tissue-specific proteins. We also successfully profiled HSP90 interactors in muscles and neurons and identified tissue-specific interactors and stress-related interactors. Our study demonstrates that MACSPI can be used to profile tissue-specific proteomes and interactomes in intact multicellular organisms.