An integrated method for single cell proteomics with simultaneous measurements of intracellular drug concentration implicates new mechanisms for adaptation to KRASG12D inhibitors.

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In the context of chemotherapeutics, these diverse responses may lead to individual adaptation mechanisms and ultimately multiple distinct methods of resistance. The obvious question from a pharmacology perspective is how intracellular concentrations of active drug varies between individual cells, and what role does that variation play in drug response heterogeneity? To date, no integrated methods for rapidly measuring intracellular drug levels while simultaneously measuring drug responses have been described. This study describes a method for single cell preparation that allows proteins to be extracted and digested from single cells while maintaining conditions for small molecules to be simultaneously measured. The method as described allows up to 40 cells to be analyzed per instrument per day. When applied to a KRASG12D small molecule inhibitor I observe a wide degree of intracellular levels of the drug, and that proteomic responses largely stratify based on the concentration of drug within each single cell. Further work is in progress to develop and standardize this method and - more importantly - to normalize drug measurements against direct measurements of cell volume. However, these preliminary results appear promising for the identification of single cells with unique drug response mechanisms. All data described in this study has been made publicly available through the ProteomeXchange consortium under accession PXD046002.", "claims": [{"id": "claim-method", "statement": "To further evaluate proteome specific alterations consistent with higher relative intracellular concentration, I first performed a simple correlation analysis between the 210 proteins at were detected in at least 50% of all cells versus the observed MRTX1133 concentrations.", "kind": "methodological", "target": null, "evidence": [{"asset": "source:pmid:38014353", "locator": "sections[14] \"Distinct proteomic alterations are observed in single cells with higher observed drug concentration\" paragraph 2", "quote": "To further evaluate proteome specific alterations consistent with higher relative intracellular concentration, I first performed a simple correlation analysis between the 210 proteins at were detected in at least 50% of all cells versus the observed MRTX1133 concentrations."}], "state": "complete"}, {"id": "claim-data-access", "statement": "All Bruker .d files, SpectroNaut processed results and Skyline files have been deposited at the ProteomeXchange MASSIVE partner repository and can be accessed as accession PXD046002.", "kind": "methodological", "target": null, "evidence": [{"asset": "source:pmid:38014353", "locator": "sections[7] \"Data Availability Statement\" paragraph 2", "quote": "All Bruker .d files, SpectroNaut processed results and Skyline files have been deposited at the ProteomeXchange MASSIVE partner repository and can be accessed as accession PXD046002."}], "state": "complete"}, {"id": "claim-target-result", "statement": "Single-cell proteomic responses largely stratified according to the intracellular concentration of the KRASG12D inhibitor MRTX1133.", "kind": "qualitative", "target": null, "evidence": [{"asset": "source:pmid:38014353", "locator": "sections[0] \"Abstract\" paragraph 1", "quote": "When applied to a KRASG12D small molecule inhibitor I observe a wide degree of intracellular levels of the drug, and that proteomic responses largely stratify based on the concentration of drug within each single cell."}], "state": "complete"}], "gaps": []}, "experiment": {"state": "partial", "objective": "Single-cell proteomic responses largely stratified according to the intracellular concentration of the KRASG12D inhibitor MRTX1133.", "steps": [{"id": "read_published_estimate", "order": 1, "name": "Read the published estimate from the source", "kind": "other", "state": "complete", "blocked_on": null, "description": "The claim, method and evidence span published with the source article.", "duration_hours": null, "parameters": {}, "inputs": ["source:pmid:38014353"], "outputs": ["the published estimate and its evidence"], "instruments": [], "reagents": []}, {"id": "obtain_source_data", "order": 2, "name": "Obtain the source data from the named repository", "kind": "data-preparation", "state": "blocked", "blocked_on": "data:accession_unresolved", "description": "The GEO series accession resolves and its sample accessions are listed. 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