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The ability to assign cellular origin to low-abundance secreted factors in extracellular vesicles (EVs) would greatly facilitate the analysis of paracrine-mediated signaling. Here, we report a method, named selective isolation of extracellular vesicles (SIEVE), which uses cell type-specific proteome labeling via stochastic orthogonal recoding of translation (SORT) to install bioorthogonal reactive groups into the proteins derived from the cells targeted for labeling. We establish the native purification of intact EVs from a target cell, via a bioorthogonal tetrazine ligation, leading to copurification of the largely unlabeled EV proteome from the same cell. SIEVE enables capture of EV proteins at levels comparable with those obtained by antibody-based methods, which capture all EVs regardless of cellular origin, and at levels 20× higher than direct capture of SORT-labeled proteins. Using proteomic analysis, we analyze nonlabeled cargo proteins of EVs and show that the enhanced sensitivity of SIEVE allows for unbiased and comprehensive analysis of EV proteins from subpopulations of cells as well as for cell-specific EV proteomics in complex coculture systems. SIEVE can be applied with high efficiency in a diverse range of existing model systems for cell-cell communication and has direct applications for cell-of-origin EV analysis and for protein biomarker discovery.

Mismatches in DNA occur naturally during replication and as a result of endogenous DNA damaging agents, but the mismatch repair (MMR) pathway acts to correct mismatches before subsequent rounds of replication. The loss of MMR carries dire consequences, including increased mutation rates, carcinogenesis, and resistance to a variety of clinical anti-cancer agents, such as cisplatin and DNA alkylators. Rhodium metalloinsertors previously developed in our laboratory bind to DNA mismatches with high affinity and specificity, and represent a promising strategy to target mismatches in cells. Thus, uncorrected mismatches can be exploited to provide a basis of discrimination between MMR-deficient, cancerous cells and MMR-proficient, healthy cells. Here we describe the application of rhodium metalloinsertors to inhibit cellular proliferation selectively in MMR-deficient cells compared to those that are MMR-proficient. The colorectal carcinoma cell lines HCT116N and HCT116O serve as an isogenic model system for MMR deficiency. We show that the Δ-isomer of an octahedral rhodium complex containing a bulky chelate ligand for insertion into a DNA mismatch is active both in targeting base mismatches in vitro and in inhibiting DNA synthesis selectively in the HCT116O cell line. A family of derivative complexes with varying ancillary ligands has also been synthesized, and both DNA mismatch binding affinities and anti-proliferative activities against the HCT116 cell lines have been determined. DNA photocleavage experiments reveal that all complexes bind to the mismatched sites with high specificities; DNA binding affinities to oligonucleotides containing single base CA and CC mismatches, obtained through photocleavage titration or competition, vary from 104 to 108 M-1 for the series of complexes. Significantly, binding affinities are found to be inversely related to ancillary ligand size and directly related to differential inhibition of the HCT116 cell lines. The observed trend in binding affinity is consistent with the metalloinsertion mode where the complex binds from the minor groove with ejection of mismatched base pairs. The correlation between binding affinity and targeting of the MMR-deficient cell line suggests that rhodium metalloinsertors exert their selective biological effects on MMR-deficient cells through mismatch binding in vivo. In particular, rhodium metalloinsertors bearing dipyridylamine ancillary ligands are shown to exhibit accelerated cellular uptake. This increased uptake allows us to observe additional cellular responses to these agents, including disruption of the cell cycle, monitored by flow cytometry assays, and induction of necrosis, monitored by dye exclusion and caspase inhibition assays, that also occur preferentially in the HCT116O cell line. Finally, these cellular responses provide insight into the mechanisms underlying the selective activity of this novel class of targeted anti-cancer agents, and are consistent with the idea that repair proteins are activated in response to DNA mismatch binding.

We develop an approach to tag proteomes synthesized by specific cell types in dissociated cortex, brain slices, and the brains of live mice. By viral-mediated expression of an orthogonal pyrrolysyl-tRNA synthetase-tRNAXXX pair in a cell type of interest and providing a non-canonical amino acid with a chemical handle, we selectively label neuronal or glial proteomes. The method enables the identification of proteins from spatially and genetically defined regions of the brain., Michael J Fox Foundation (MJFF) (unknown), Parkinson's UK (None), Cure Parkinson's Trust (unknown)