Determination of phosphorylation sites in the DivIVA cytoskeletal protein of Streptomyces coelicolor by targeted LC-MS/MS.

{"sci_version": "1.2", "schema": "https://corpus.bntrg.co.uk/sci/1.0/sci.schema.json", "id": "pmid:23905541", "kind": "science-record", "revision": 6, "content_hash": "sha256:fdbdbd135841d12fc6d5de635cdb7d6dc3e9eb6fa124ccec8f1244de9c8a2858", "created_at": "2026-11-01T15:40:03Z", "updated_at": "2026-11-01T21:19:42Z", "generator": {"name": "corpus-assay-engine", "run_id": "reading-sci-2026-11-01"}, "paper": {"state": "complete", "title": "Determination of phosphorylation sites in the DivIVA cytoskeletal protein of Streptomyces coelicolor by targeted LC-MS/MS.", "doi": "10.1021/pr400524d", "pmid": "23905541", "authors": ["Gerhard Saalbach", "Antje M Hempel", "Marielle Vigouroux", "Klas Flärdh", "Mark J Buttner", "Michael J Naldrett"], "journal": "Journal of proteome research", "year": 2013, "abstract": "The filamentous bacterium Streptomyces coelicolor modulates polar growth and branching by phosphorylating the cytoskeletal protein DivIVA. Previous MALDI-TOF analysis of DivIVA showed that a large 7.2 kDa tryptic peptide was multiply phosphorylated. To aid localization of the phosphorylation sites, we introduced additional tryptic cleavage sites into DivIVA, and the resulting phosphopeptides were analyzed by LC-MS/MS. Phosphopeptide isomers could be separated chromatographically, but because of overlapping elution and spectrum quality, site assignment by standard software tools was ambiguous. Because fragment ions carrying the phosphate group are essential for confident localization, large numbers of spectra were collected using targeted LC-MS/MS, and a special script was developed for plotting the elution of site-determining fragments from those spectra under the XIC of the parent ions. Where multiple phosphopeptide isomers were present, the elution of the site-determining y-ions perfectly coincided with the elution of the corresponding phosphopeptide isomer. This method represents a useful tool for user inspection of spectra derived from phosphopeptide isomers and significantly increases confidence when defining phosphorylation sites. In this way, we show that DivIVA is phosphorylated in vivo on five sites in the C-terminal part of the protein (T304, S309, S338, S344, and S355). The data have been deposited to the ProteomeXchange Consortium with identifier PXD000095.", "claims": [{"id": "claim-method", "statement": "further into ScaffoldPTM 2.0 (Proteome Software) for evaluation of the phosphorylation sites using the Ascore4 probabilistic algorithm and scoring technique incorporated into ScaffoldPTM", "kind": "methodological", "target": null, "evidence": [{"asset": "source:pmid:23905541", "locator": "sections[9] \"Software Tool for Visualization of Site-Determining MS2 ions from Targeted LC–MS/MS\" paragraph 2", "quote": "further into ScaffoldPTM 2.0 (Proteome Software) for evaluation of the phosphorylation sites using the Ascore4 probabilistic algorithm and scoring technique incorporated into ScaffoldPTM"}], "state": "complete"}], "gaps": []}, "experiment": {"state": "partial", "objective": null, "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:23905541"], "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_absent", "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": [], "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": [], "outputs": ["the replicated result and its comparison"], "instruments": [], "reagents": []}], "materials": [], "instruments": [], "reagents": [], "conditions": {}, "gaps": ["step:obtain_source_data blocked on data:accession_absent"]}, "data": {"state": "partial", "assets": [{"id": "source:pmid:23905541", "role": "manuscript", "format": "xml", "media_type": "application/xml", "uri": "https://doi.org/10.1021/pr400524d", "sha256": "sha256:6e5150f27cf09d84549de207ed5e7ce12fd603e7a8370022ee1de93a2e484a47", "bytes": 28710, "access": "open", "state": "resolved", "reader": null, "reader_status": "reader_required", "identifier": "10.1021/pr400524d", "version": null, "note": "Full article fetched from PMC; the checksum covers the rendered section body, not the original XML bytes.", "structure": {"pmcid": "PMC3787806", "source": "europe_pmc_fulltext", "coverage": "whole_body", "text_hash_scope": "rendered_section_body"}}, {"id": "asset:table:pmid:23905541:1", "role": "table", "format": "jats-table", "media_type": "application/vnd.jats.table+json", "uri": null, "sha256": "sha256:376d8588fd98ca5735781caede3e53d94917c7101790695cf0eada1b9777f87a", "bytes": 878, "access": "open", "state": "verified", "reader": "read_table", "reader_status": "available", "identifier": "section[10]/table-wrap[1]#tbl1", "version": null, "note": "Comparison of Intensities and Retention Times (RT, min) of Nonphosphorylated Peptides and Their Phosphorylated Counterparts (Isomers)", "structure": {"pmcid": "PMC3787806", "section_index": 10, "section_title": "Unphosphorylated and Phosphorylated Phosphopeptide Isomers", "row_count": 9, "column_count": 5}, "table": {"label": "Table 1", "caption": "Comparison of Intensities and Retention Times (RT, min) of Nonphosphorylated Peptides and Their Phosphorylated Counterparts (Isomers)", "rows": [["peptide", "phosphopeptide isomer Sequence", "intensitya", "ratio (P)", "RTa"], ["Peptide 1", "QLETQADDSLAPPR", "4.63 × 109", "", "28.31"], ["Peptide 1", "QLETQADDpSLAPPR", "2.64 × 107", "0.005699", "28.03"], ["Peptide 1", "QLEpTQADDSLAPPR", "2.67 × 106", "0.000577", "31.27"], ["Peptide 2", "TPATASLPPSPAPSMAPAGASAPSYGGNR", "1.92 × 107", "", "33.02"], ["Peptide 2", "TPATASLPPSPAPSMAPAGASAPpSYGGNR", "7.28 × 106", "0.37996", "33.63"], ["Peptide 3", "SMGGGPGQSGPSYGGQR", "7.91 × 108", "", "22.04"], ["Peptide 3", "SMGGGPGQSGPpSYGGQR", "3.82 × 108", "0.48319", "22.80"], ["Peptide 3", "pSMGGGPGQSGPSYGGQR", "nd", "0.06b", "23.25b"]], "row_count": 9, "column_count": 5, "numeric_cells": 28, "header_rows": 1, "locator": "section[10]/table-wrap[1]#tbl1"}}], "gaps": []}, "analysis": {"state": "partial", "methods": [{"id": "method-1", "family": null, "specific": null, "label": "Ascore probabilistic algorithm", "dsl_operations": [], "missing_operations": [], "expressible": null, "parameters": {}, "data_shapes": [], "evidence": {"score": 1.0, "band": "verbatim", "quote": "further into ScaffoldPTM 2.0 (Proteome Software) for evaluation of the phosphorylation sites using the Ascore4 probabilistic algorithm and scoring technique incorporated into ScaffoldPTM"}}], "targets": [], "gaps": ["method.reader_named_unmapped"]}, "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": "data", "blocked_on": "data:accession_absent", "axes": {"method": {"tier": "R4", "expressible": false, "required_operations": [], "missing_operations": [], "basis": "The reader quoted the method the paper names (Ascore probabilistic algorithm), 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": "R4", "reachable": false, "accessions": [], "resolved_accessions": [], "statements": [], "basis": "The paper names no accession to its source data, so the data has to come from the authors."}}, "can_run": {"dsl_operations": [], "assets": ["source:pmid:23905541", "asset:table:pmid:23905541:1"], "steps": ["run_analysis"]}, "must_build": {"dsl_operations": [], "converters": [], "readers": [], "capabilities": [], "data_gaps": ["data:geo:None"]}, "gaps": ["data:accession_absent"]}, "review": {"state": "partial", "machine_critique": {"kind": "extraction_review", "reader": {"model": "MiniMax-M3.1-Flash", "provider": "minimax", "provider_family": "minimax", "read_at": null, "independence": "same_provider"}, "evidence_check": {"method": "verbatim", "availability": null}, "calls": 0, "usage": {}}, "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:23905541", "type": "paper", "label": "pmid:23905541"}, {"id": "source:pmid:23905541", "type": "manuscript", "label": "PMID 23905541 full text"}, {"id": "data:geo:None", "type": "dataset", "label": "GEO None"}], "activities": [{"id": "activity-reading-sci-2026-11-01", "type": "classification", "used": [{"id": "pmid:23905541", "type": "paper", "label": "pmid:23905541"}, {"id": "source:pmid:23905541", "type": "manuscript", "label": "PMID 23905541 full text"}, {"id": "data:geo:None", "type": "dataset", "label": "GEO None"}], "agent": "agent:corpus-assay-engine"}, {"id": "activity-read-23905541", "type": "extraction", "used": ["source:pmid:23905541"], "agent": "agent:minimax-reader"}, {"id": "activity-resolve-None", "type": "accession_resolution", "used": ["data:geo:None"], "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:23905541", "parent_hash": "sha256:dc4a6fc409b111a13851f096cc8326fad5494621ce48eea81fbf92eb5413fec0", "fork_of": null, "generation": 5}, "gaps": ["execution.not_run", "method.reader_named_unmapped", "review.human_critique", "review.independent_reader", "review.machine_critique", "step:obtain_source_data blocked on data:accession_absent"], "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": ["otein of Streptomyces coelicolor by targeted LC-MS/MS.", "e resulting phosphopeptides were analyzed by LC-MS/MS. Phosphopeptide isomers could be separated c", "ers of spectra were collected using targeted LC-MS/MS, and a special script was developed for plot", "Experimental Section Mass Spectrometric Analysis of Phosphorylation Sites Protein sa"], "order_basis": "paper_text_order:68", "id": "assay:1"}, {"role": "data_generation", "definition_id": "animal_model", "definition_version": "99e1195c7d6f", "definition_digest": null, "required_operations": [], "support_state": "partial", "source_hashes": {"assay_lexicon": "99e1195c7d6f"}, "confidence": 0.77, "evidence": ["s way, we show that DivIVA is phosphorylated in vivo on five sites in the C-terminal part of the"], "order_basis": "paper_text_order:1386", "id": "assay:2"}]}