Single Nucleotide Resolution RNA-Protein Cross-Linking Mass Spectrometry: A Simple Extension of the CLIR-MS Workflow.

{"sci_version": "1.2", "schema": "https://corpus.bntrg.co.uk/sci/1.0/sci.schema.json", "id": "pmid:34714631", "kind": "science-record", "revision": 2, "content_hash": "sha256:0991b6754ce2c588a55461b3ed7fc0287bb471581023e3902b573a095a2420fd", "created_at": "2026-11-01T17:36:23Z", "updated_at": "2026-11-01T18:57:17Z", "generator": {"name": "corpus-assay-engine", "run_id": "reading-sci-2026-11-01"}, "paper": {"state": "complete", "title": "Single Nucleotide Resolution RNA-Protein Cross-Linking Mass Spectrometry: A Simple Extension of the CLIR-MS Workflow.", "doi": "10.1021/acs.analchem.1c02384", "pmid": "34714631", "authors": ["Michael Götze", "Chris P Sarnowski", "Tebbe de Vries", "Anna Knörlein", "Frédéric H-T Allain", "Jonathan Hall", "Ruedi Aebersold", "Alexander Leitner"], "journal": "Analytical chemistry", "year": 2021, "abstract": "RNA-protein interactions mediate many intracellular processes. CLIR-MS (cross-linking of isotope-labeled RNA and tandem mass spectrometry) allows the identification of RNA-protein interaction sites at single nucleotide/amino acid resolution in a single experiment. Using isotopically labeled RNA segments for UV-light-induced cross-linking generates characteristic isotope patterns that constrain the sequence database searches, increasing spatial resolution. Whereas the use of segmentally isotopically labeled RNA is effective, it is technically involved and not applicable in some settings, e.g., in cell or tissue samples. Here we introduce an extension of the CLIR-MS workflow that uses unlabeled RNA during cross-linking and subsequently adds an isotopic label during sample preparation for MS analysis. After RNase and protease digests of a cross-linked complex, the nucleic acid part of a peptide-RNA conjugate is labeled using the enzyme T4 polynucleotide kinase and a 1:1 mixture of heavy 18O4-γ-ATP and light ATP. In this simple, one-step reaction, three heavy oxygen atoms are transferred from the γ-phosphate to the 5'-end of the RNA, introducing an isotopic shift of 6.01 Da that is detectable by mass spectrometry. We applied this approach to the RNA recognition motif (RRM) of the protein FOX1 in complex with its cognate binding substrate, FOX-binding element (FBE) RNA. We also labeled a single phosphate within an RNA and unambiguously determined the cross-linking site of the FOX1-RRM binding to FBE at single residue resolution on the RNA and protein level and used differential ATP labeling for relative quantification based on isotope dilution. Data are available via ProteomeXchange with the identifier PXD024010.", "claims": [{"id": "claim-data-access", "statement": "Data are available via ProteomeXchange with the identifier PXD024010.", "kind": "methodological", "target": null, "evidence": [{"asset": "source:pmid:34714631", "locator": "sections[0] \"Abstract\" paragraph 1", "quote": "Data are available via ProteomeXchange with the identifier PXD024010."}], "state": "complete"}, {"id": "claim-target-result", "statement": "We also labeled a single phosphate within an RNA and unambiguously determined the cross-linking site of the FOX1-RRM binding to FBE at single residue resolution on the RNA and protein level and used differential ATP labeling for relative quantification based on isotope dilution.", "kind": "qualitative", "target": null, "evidence": [{"asset": "source:pmid:34714631", "locator": "sections[0] \"Abstract\" paragraph 1", "quote": "We also labeled a single phosphate within an RNA and unambiguously determined the cross-linking site of the FOX1-RRM binding to FBE at single residue resolution on the RNA and protein level and used differential ATP labeling for relative quantification based on isotope dilution."}], "state": "complete"}], "gaps": []}, "experiment": {"state": "complete", "objective": "We also labeled a single phosphate within an RNA and unambiguously determined the cross-linking site of the FOX1-RRM binding to FBE at single residue resolution on the RNA and protein level and used differential ATP labeling for relative quantification based on isotope dilution.", "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:34714631"], "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": "complete", "blocked_on": null, "description": "The GEO series accession resolves and its sample accessions are listed. This lane did not download the dataset or read its licence.", "duration_hours": null, "parameters": {}, "inputs": ["data:geo:PXD024010"], "outputs": ["the source data with its repository provenance"], "instruments": [], "reagents": []}, {"id": "run_analysis", "order": 3, "name": "Run the published analysis", "kind": "analysis", "state": "complete", "blocked_on": null, "description": "Apply the paper's statistical method to the resolved series and compare the result with the published target.", "duration_hours": null, "parameters": {}, "inputs": ["data:geo:PXD024010"], "outputs": ["the replicated result and its comparison"], "instruments": [], "reagents": []}], "materials": [], "instruments": [], "reagents": [], "conditions": {}, "gaps": []}, "data": {"state": "partial", "assets": [{"id": "source:pmid:34714631", "role": "manuscript", "format": "xml", "media_type": "application/xml", "uri": "https://doi.org/10.1021/acs.analchem.1c02384", "sha256": "sha256:e8cc4bf0be640bee4ca910bfe51fcd442ad1551f286553011ef880361b2ab33c", "bytes": 48346, "access": "open", "state": "resolved", "reader": null, "reader_status": "reader_required", "identifier": "10.1021/acs.analchem.1c02384", "version": null, "note": "Full article fetched from PMC; the checksum covers the rendered section body, not the original XML bytes.", "structure": {"pmcid": "PMC8581962", "source": "europe_pmc_fulltext", "coverage": "whole_body", "text_hash_scope": "rendered_section_body"}}, {"id": "data:geo:PXD024010", "role": "raw", "format": "geo-series", "media_type": "application/vnd.ncbi.geo-series+json", "uri": "https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=PXD024010", "sha256": null, "bytes": null, "access": "open", "state": "resolved", "reader": null, "reader_status": "reader_required", "identifier": "PXD024010", "version": null, "note": "Data are available via ProteomeXchange with the identifier PXD024010.", "structure": {"repository": "GEO", "access_route": "GEO accession URL", "access_statement_verbatim": "Data are available via ProteomeXchange with the identifier PXD024010.", "access_statement_kind": "data_availability_statement", "sample_accessions": [], "sample_count": null, "group_sizes": null, "group_sizes_basis": null, "platform": null, "taxon": null, "title": "Single Nucleotide Resolution RNA-Protein Cross-Linking/MS: Simple Extension of the CLIR-MS Workflow", "resolution_checked_at": "2026-11-01T17:30:50Z", "resolution_check": "identifier_resolution_only_no_download_no_licence_read", "downloaded": false, "licence_read": false}}], "gaps": ["asset:data:geo:PXD024010 not downloaded", "asset:data:geo:PXD024010 licence not read"]}, "analysis": {"state": "absent", "methods": [], "targets": [], "gaps": ["method", "operations"]}, "capability": {"state": "complete", "requirements": [{"capability_id": "capability:computational_analysis", "label": "Computational analysis platform", "assay_families": ["meta_analysis", "machine_learning", "survival_analysis", "statistical_regression", "pca", "molecular_docking", "network_analysis", "differential_expression", "gwas"], "availability_state": "owned", "tier_unlocked": "R1", "candidate": "Automated Assays Laravel application and simulated adapters"}], "gaps": []}, "readiness": {"state": "blocked", "tier": "R4", "blocking_axis": "method", "blocked_on": "method:not_determinable", "axes": {"method": {"tier": "R4", "expressible": false, "required_operations": [], "missing_operations": [], "basis": "The reader quoted the method the paper names (not named), but the corpus's method extractor could not map it onto a DSL operation, so the method is published but not expressible."}, "capability": {"tier": "R1", "capability_id": "capability:computational_analysis", "availability_state": "owned", "basis": "The computational analysis is owned. The physical bench the paper used is not, so a physical validation cannot be run here."}, "data": {"tier": "R1", "reachable": true, "accessions": ["PXD024010"], "resolved_accessions": ["PXD024010"], "statements": ["Data are available via ProteomeXchange with the identifier PXD024010."], "basis": "The paper names PXD024010 in ProteomeXchange Consortium via the PRIDE partner repository and the accession resolves in the repository. No dataset was downloaded and no licence was read."}}, "can_run": {"dsl_operations": [], "assets": ["source:pmid:34714631", "data:geo:PXD024010"], "steps": ["run_analysis"]}, "must_build": {"dsl_operations": [], "converters": [], "readers": [], "capabilities": [], "data_gaps": ["data:geo:PXD024010"]}, "gaps": ["method:not_determinable"]}, "review": {"state": "partial", "machine_critique": {"kind": "extraction_review", "reader": {"model": "MiniMax-M3.1-Flash", "provider": "minimax", "provider_family": "minimax", "read_at": "2026-11-01T17:05:15Z", "independence": "single_reader"}, "evidence_check": {"method": null, "availability": "verbatim"}, "calls": 5, "usage": {"prompt_tokens": 14108, "completion_tokens": 935, "cached_tokens": 128}}, "human_critique": null, "stages": [{"id": "extraction", "state": "complete", "reader": "MiniMax-M3.1-Flash", "provider": "minimax"}], "gaps": ["review.independent_reader", "review.machine_critique", "review.human_critique"]}, "execution": {"state": "absent", "status": "not_run", "mode": null, "runs": [], "gaps": ["execution.not_run"]}, "provenance": {"entities": [{"id": "pmid:34714631", "type": "paper", "label": "pmid:34714631"}, {"id": "source:pmid:34714631", "type": "manuscript", "label": "PMID 34714631 full text"}, {"id": "data:geo:PXD024010", "type": "dataset", "label": "GEO PXD024010"}], "activities": [{"id": "activity-reading-sci-2026-11-01", "type": "classification", "used": [{"id": "pmid:34714631", "type": "paper", "label": "pmid:34714631"}, {"id": "source:pmid:34714631", "type": "manuscript", "label": "PMID 34714631 full text"}, {"id": "data:geo:PXD024010", "type": "dataset", "label": "GEO PXD024010"}], "agent": "agent:corpus-assay-engine"}, {"id": "activity-read-34714631", "type": "extraction", "used": ["source:pmid:34714631"], "agent": "agent:minimax-reader"}, {"id": "activity-resolve-PXD024010", "type": "accession_resolution", "used": ["data:geo:PXD024010"], "agent": "agent:corpus-assay-engine"}], "agents": [{"id": "agent:corpus-assay-engine", "type": "software", "label": "Corpus assay engine"}, {"id": "agent:minimax-reader", "type": "software", "label": "MiniMax literature reader"}], "source_run_id": "reading-sci-2026-11-01", "dsl_catalogue_fingerprint": "343fb30756ba6ec3", "capability_table_fingerprint": "2cb3e42f047d2ecf", "snapshot_id": "ba34b49490d9bfaf"}, "lineage": {"parent_id": "pmid:34714631", "parent_hash": "sha256:325926a9541a78b4bff95d95f2d217263745032e5dcfcf102334a63af1aecbc0", "fork_of": null, "generation": 1}, "gaps": ["asset:data:geo:PXD024010 licence not read", "asset:data:geo:PXD024010 not downloaded", "execution.not_run", "method", "operations", "review.human_critique", "review.independent_reader", "review.machine_critique"], "assays": [{"role": "data_generation", "definition_id": "mass_spectrometry", "definition_version": "99e1195c7d6f", "definition_digest": null, "required_operations": [], "support_state": "partial", "source_hashes": {"assay_lexicon": "99e1195c7d6f"}, "confidence": 0.99, "evidence": ["leotide Resolution RNA-Protein Cross-Linking Mass Spectrometry: A Simple Extension of the CLIR-MS Workflow.", "ss-linking of isotope-labeled RNA and tandem mass spectrometry) allows the identification of RNA-protein in", "topic shift of 6.01 Da that is detectable by mass spectrometry. We applied this approach to the RNA recogni", "chment, the sample was analyzed by LC–MS/MS. Mass spectrometric analysis revealed multiple isotopic pairs in"], "order_basis": "paper_text_order:10", "id": "assay:1"}]}