Tagging and Enriching Proteins Enables Cell-Specific Proteomics

Abstract

SummaryCell-specific proteomics in multicellular systems and whole animals is a promising approach to understand the differentiated functions of cells and tissues. Here, we extend our stochastic orthogonal recoding of translation (SORT) approach for the co-translational tagging of proteomes with a cyclopropene-containing amino acid in response to diverse codons in genetically targeted cells, and create a tetrazine-biotin probe containing a cleavable linker that offers a way to enrich and identify tagged proteins. We demonstrate that SORT with enrichment, SORT-E, efficiently recovers and enriches SORT tagged proteins and enables specific identification of enriched proteins via mass spectrometry, including low-abundance proteins. We show that tagging at distinct codons enriches overlapping, but distinct sets of proteins, suggesting that tagging at more than one codon enhances proteome coverage. Using SORT-E, we accomplish cell-specific proteomics in the fly. These results suggest that SORT-E will enable the definition of cell-specific proteomes in animals during development, disease progression, and learning and memory.

Inverse electron demand Diels–Alder reactions in chemical biology

“…155 In general, the cyclopropene handle has the advantage of being efficiently incorporated into proteins without suffering from isomerisation and presenting a relatively small size and fast kinetics (labelling reaction on proteins completed within 30 min). 155,156 copper-free dibenzocyclooctyne-azide cycloaddition. In comparison to strained alkenes, unstrained alkenes provide some advantages such as their small size, easy synthesis and enhanced stability towards cellular nucleophiles.…”

“…The beads can be washed and enriched proteins may be detected using MS after reductive cleavage of the diazobenzene linker. 156…”

Bifunctional Non-Canonical Amino Acids: Combining Photo-Crosslinking with Click Chemistry

“…Methods such as stochastic orthogonal recoding of translation (SORT) [45] have been developed to introduce ncAAs into the whole proteome simultaneously. Instead of suppressing a stop codon, the genome is expanded by stochastically incorporating ncAAs in place of certain sense codons and under competition with the native amino acids [46]. Combining this with photo-crosslinkers has the potential to capture interactions throughout the whole proteome, as has been shown by the surrogate-based method, where a photo-crosslinker analog of a canonic amino acid is supplemented and incorporated residue specifically [47,48].…”

Cell-selective proteomics for biological discovery

“…OP-Puro requires an azide-based affinity handle or resin for enrichment [43]. SORT uses cyclopropene labels and tetrazine linkers in a ligation reaction reported to be 100 to 1000 times faster than the strain-promoted azide-alkyne cycloaddition [41]. BONCAT labels with either alkynes or azides, and enriches with complementary azide or alkyne reagents.…”

“…Elliott et al . have characterized the enrichment process and found that tagging at different codons leads to the enrichment of overlapping, but distinct sets of proteins [41]. The authors noted that simultaneous expression of multiple tRNAs (i.e., tRNA-Ala, -Ser and -Met) increases labeling efficiency.…”