Assessment of current mass spectrometric workflows for the quantification of low abundant proteins and phosphorylation sites.

{"sci_version": "1.2", "schema": "https://corpus.bntrg.co.uk/sci/1.0/sci.schema.json", "id": "pmid:26550600", "kind": "science-record", "revision": 6, "content_hash": "sha256:f4497dd20db5380a2763d16df6149b63560f04219c74f2fb31d3f46d6fedf472", "created_at": "2026-11-01T15:40:13Z", "updated_at": "2026-11-01T21:20:17Z", "generator": {"name": "corpus-assay-engine", "run_id": "reading-sci-2026-11-01"}, "paper": {"state": "complete", "title": "Assessment of current mass spectrometric workflows for the quantification of low abundant proteins and phosphorylation sites.", "doi": "10.1016/j.dib.2015.08.015", "pmid": "26550600", "authors": ["Manuel Bauer", "Erik Ahrné", "Anna P Baron", "Timo Glatter", "Luca L Fava", "Anna Santamaria", "Erich A Nigg", "Alexander Schmidt"], "journal": "Data in brief", "year": 2015, "abstract": "The data described here provide a systematic performance evaluation of popular data-dependent (DDA) and independent (DIA) mass spectrometric (MS) workflows currently used in quantitative proteomics. We assessed the limits of identification, quantification and detection for each method by analyzing a dilution series of 20 unmodified and 10 phosphorylated synthetic heavy labeled reference peptides, respectively, covering six orders of magnitude in peptide concentration with and without a complex human cell digest background. We found that all methods performed very similarly in the absence of background proteins, however, when analyzing whole cell lysates, targeted methods were at least 5-10 times more sensitive than directed or DDA methods. In particular, higher stage fragmentation (MS3) of the neutral loss peak using a linear ion trap increased dynamic quantification range of some phosphopeptides up to 100-fold. We illustrate the power of this targeted MS3 approach for phosphopeptide monitoring by successfully quantifying 9 phosphorylation sites of the kinetochore and spindle assembly checkpoint component Mad1 over different cell cycle states from non-enriched pull-down samples. The data are associated to the research article 'Evaluation of data-dependent and data-independent mass spectrometric workflows for sensitive quantification of proteins and phosphorylation sites׳ (Bauer et al., 2014) [1]. The mass spectrometry and the analysis dataset have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE partner repository with the dataset identifier PXD000964.", "claims": [{"id": "claim-method", "statement": "Finally, we generated dilution profile correlations and applied an established algorithm [3] to determine LOQ and LOD values as well as linear correlations (Pearson׳s correlation coefficient (R2) from highest concentration to LOQ) for each MS-method and peptide analyzed.", "kind": "methodological", "target": null, "evidence": [{"asset": "source:pmid:26550600", "locator": "sections[13] \"Peptide quantification/determination of quantification limits\" paragraph 2", "quote": "Finally, we generated dilution profile correlations and applied an established algorithm [3] to determine LOQ and LOD values as well as linear correlations (Pearson׳s correlation coefficient (R2) from highest concentration to LOQ) for each MS-method and peptide analyzed."}], "state": "complete"}, {"id": "claim-data-access", "statement": "The mass spectrometry and the analysis dataset have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE partner repository with the dataset identifier PXD000964.", "kind": "methodological", "target": null, "evidence": [{"asset": "source:pmid:26550600", "locator": "sections[0] \"Abstract\" paragraph 1", "quote": "The mass spectrometry and the analysis dataset have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE partner repository with the dataset identifier PXD000964."}], "state": "complete"}, {"id": "claim-target-result", "statement": "The study found that targeted methods were at least 5–10 times more sensitive than directed or DDA methods in whole cell lysates, using an established algorithm to determine LOQ/LOD values and Pearson’s correlation coefficient (R2) for dilution profiles; the source data are available through ProteomeXchange and PRIDE under identifier PXD000964.", "kind": "quantitative", "target": {"value": 10.0, "unit": "fold change", "tolerance": null, "source": "We found that all methods performed very similarly in the absence of background proteins, however, when analyzing whole cell lysates, targeted methods were at least 5–10 times more sensitive than directed or DDA methods."}, "evidence": [{"asset": "source:pmid:26550600", "locator": "sections[0] \"Abstract\" paragraph 1", "quote": "We found that all methods performed very similarly in the absence of background proteins, however, when analyzing whole cell lysates, targeted methods were at least 5–10 times more sensitive than directed or DDA methods."}, {"asset": "source:pmid:26550600", "locator": "sections[13] \"Peptide quantification/determination of quantification limits\" paragraph 2", "quote": "Finally, we generated dilution profile correlations and applied an established algorithm [3] to determine LOQ and LOD values as well as linear correlations (Pearson׳s correlation coefficient (R2) from highest concentration to LOQ) for each MS-method and peptide analyzed."}, {"asset": "source:pmid:26550600", "locator": "sections[0] \"Abstract\" paragraph 1", "quote": "The mass spectrometry and the analysis dataset have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE partner repository with the dataset identifier PXD000964."}], "state": "complete"}], "gaps": []}, "experiment": {"state": "complete", "objective": "The study found that targeted methods were at least 5–10 times more sensitive than directed or DDA methods in whole cell lysates, using an established algorithm to determine LOQ/LOD values and Pearson’s correlation coefficient (R2) for dilution profiles; the source data are available through ProteomeXchange and PRIDE under identifier PXD000964.", "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:26550600"], "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:PXD000964"], "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:PXD000964"], "outputs": ["the replicated result and its comparison"], "instruments": [], "reagents": []}], "materials": [], "instruments": [], "reagents": [], "conditions": {}, "gaps": []}, "data": {"state": "partial", "assets": [{"id": "source:pmid:26550600", "role": "manuscript", "format": "xml", "media_type": "application/xml", "uri": "https://doi.org/10.1016/j.dib.2015.08.015", "sha256": "sha256:92a095e0614fdcc15d292d4de1e970e6ed4e31f71522f2976a9f83e92001c83b", "bytes": 25384, "access": "open", "state": "resolved", "reader": null, "reader_status": "reader_required", "identifier": "10.1016/j.dib.2015.08.015", "version": null, "note": "Full article fetched from PMC; the checksum covers the rendered section body, not the original XML bytes.", "structure": {"pmcid": "PMC4596922", "source": "europe_pmc_fulltext", "coverage": "whole_body_without_references", "text_hash_scope": "rendered_section_body"}}, {"id": "data:geo:PXD000964", "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=PXD000964", "sha256": null, "bytes": null, "access": "open", "state": "resolved", "reader": null, "reader_status": "reader_required", "identifier": "PXD000964", "version": null, "note": "The mass spectrometry and the analysis dataset have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE partner repository with the dataset identifier PXD000964.", "structure": {"repository": "GEO", "access_route": "GEO accession URL", "access_statement_verbatim": "The mass spectrometry and the analysis dataset have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE partner repository with the dataset identifier PXD000964.", "access_statement_kind": "data_availability_statement", "sample_accessions": [], "sample_count": null, "group_sizes": null, "group_sizes_basis": null, "platform": null, "taxon": null, "title": "MS approach assessment and Mad1 phosphopeptide analysis", "resolution_checked_at": "2026-10-31T18:35:55Z", "resolution_check": "identifier_resolution_only_no_download_no_licence_read", "downloaded": false, "licence_read": false}}, {"id": "asset:table:pmid:26550600:1", "role": "table", "format": "jats-table", "media_type": "application/vnd.jats.table+json", "uri": null, "sha256": "sha256:27e4611b8ce6874dd34dadfdfaa8ea5a581fd0d747e9077939974d61c0284298", "bytes": 1470, "access": "open", "state": "verified", "reader": "read_table", "reader_status": "available", "identifier": "section[1]/table-wrap[1]#t0010", "version": null, "note": null, "structure": {"pmcid": "PMC4596922", "section_index": 1, "section_title": "Specifications table", "row_count": 9, "column_count": 2}, "table": {"label": null, "caption": null, "rows": [["Subject area", "Biology, chemistry"], ["More specific subject area", "Phosphoproteomics and mass spectrometry (MS)"], ["Type of data", "MS-data, Tab-delimited and Microsoft Excel tables"], ["How data was acquired", "Easy-nLC liquid chromatography system coupled to either a LTQ Orbitrap Velos or a TSQ Vantage mass spectrometer (all Thermo Scientific)"], ["Data format", "Raw (.raw), mgf peak lists (.mgf), transition lists and Skyline processed files (.csv and.xlsx)"], ["Experimental factors", "Sensitivity assessment of different LC–MS approaches using dilution series experiments and application of the most sensitive MS method to quantify MAD1 phosphorylation sites during cell cycle."], ["Experimental features", "Different dilution series of peptides were analyzed using one dimensional liquid chromatography separation and different data-dependent and independent mass spectrometry workflows to assess their limits of quantification. The most sensitive method was applied to monitor phosphorylation site changes in immunopurified MAD1 across different cell cycle stages using thymidine and nocodazole treatment."], ["Data source location", "Basel, Switzerland"], ["Data accessibility", "The data are available via ProteomeXchange with identifier PXD000964 http://proteomecentral.proteomexchange.org/cgi/GetDataset?ID=PXD000964"]], "row_count": 9, "column_count": 2, "numeric_cells": 1, "header_rows": 0, "locator": "section[1]/table-wrap[1]#t0010"}}], "gaps": ["asset:data:geo:PXD000964 not downloaded", "asset:data:geo:PXD000964 licence not read"]}, "analysis": {"state": "partial", "methods": [{"id": "method-1", "family": null, "specific": null, "label": "Pearson’s correlation coefficient (R²); source data are available through ProteomeXchange via the PRIDE partner repository under identifier PXD000964.", "dsl_operations": [], "missing_operations": [], "expressible": null, "parameters": {}, "data_shapes": [], "evidence": {"score": 1.0, "band": "verbatim", "quote": "Finally, we generated dilution profile correlations and applied an established algorithm [3] to determine LOQ and LOD values as well as linear correlations (Pearson׳s correlation coefficient (R2) from highest concentration to LOQ) for each MS-method and peptide analyzed."}}], "targets": [{"id": "target-published-estimate", "claim_id": "claim-target-result", "operation": null, "expected_value": 10.0, "unit": "fold change", "tolerance": null, "state": "complete", "note": "The published estimate as the reader quoted it from the paper. No tolerance is published because the paper states none."}, {"id": "target-published-estimate-correlation-coefficient", "claim_id": "claim-target-result", "operation": null, "expected_value": 2.0, "unit": "correlation coefficient", "tolerance": null, "state": "complete", "note": "The published estimate as the reader quoted it from the paper. No tolerance is published because the paper states none."}], "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": "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 (Pearson’s correlation coefficient (R²); source data are available through ProteomeXchange via the PRIDE partner repository under identifier PXD000964.), 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": ["PXD000964"], "resolved_accessions": ["PXD000964"], "statements": ["The mass spectrometry and the analysis dataset have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE partner repository with the dataset identifier PXD000964."], "basis": "The paper names PXD000964 in ProteomeXchange Consortium (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:26550600", "data:geo:PXD000964", "asset:table:pmid:26550600:1"], "steps": ["run_analysis"]}, "must_build": {"dsl_operations": [], "converters": [], "readers": [], "capabilities": [], "data_gaps": ["data:geo:PXD000964"]}, "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": "2026-10-31T18:26:19Z", "independence": "same_provider"}, "evidence_check": {"method": "verbatim", "availability": "verbatim"}, "calls": 8, "usage": {"prompt_tokens": 53085, "completion_tokens": 4728, "cached_tokens": 2096}}, "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:26550600", "type": "paper", "label": "pmid:26550600"}, {"id": "source:pmid:26550600", "type": "manuscript", "label": "PMID 26550600 full text"}, {"id": "data:geo:PXD000964", "type": "dataset", "label": "GEO PXD000964"}], "activities": [{"id": "activity-reading-sci-2026-11-01", "type": "classification", "used": [{"id": "pmid:26550600", "type": "paper", "label": "pmid:26550600"}, {"id": "source:pmid:26550600", "type": "manuscript", "label": "PMID 26550600 full text"}, {"id": "data:geo:PXD000964", "type": "dataset", "label": "GEO PXD000964"}], "agent": "agent:corpus-assay-engine"}, {"id": "activity-read-26550600", "type": "extraction", "used": ["source:pmid:26550600"], "agent": "agent:minimax-reader"}, {"id": "activity-resolve-PXD000964", "type": "accession_resolution", "used": ["data:geo:PXD000964"], "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:26550600", "parent_hash": "sha256:807a8ea686b1ce79262cef3a04f49662076a419a219dead4376d380deda46142", "fork_of": null, "generation": 5}, "gaps": ["asset:data:geo:PXD000964 licence not read", "asset:data:geo:PXD000964 not downloaded", "execution.not_run", "method.reader_named_unmapped", "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": ["Assessment of current mass spectrometric workflows for the quantification of low abun", "r data-dependent (DDA) and independent (DIA) mass spectrometric (MS) workflows currently used in quantitativ", "ation of data-dependent and data-independent mass spectrometric workflows for sensitive quantification of pr", "ylation sites׳ (Bauer et al., 2014) [1]. The mass spectrometry and the analysis dataset have been deposited"], "order_basis": "paper_text_order:0", "id": "assay:1"}, {"role": "data_generation", "definition_id": "cell_culture", "definition_version": "99e1195c7d6f", "definition_digest": null, "required_operations": [], "support_state": "partial", "source_hashes": {"assay_lexicon": "99e1195c7d6f"}, "confidence": 0.99, "evidence": ["reparation of human cell digest HeLa S3 were cultured in Dulbecco׳s modified Eagle׳s medium (DMEM, In", "and sample preparation of human cell digest HeLa S3 were cultured in Dulbecco׳s modified Eagl", "lture, synchronization and kinase inhibitors HeLa S3 were cultured as described above. Cell cy"], "order_basis": "paper_text_order:4579", "id": "assay:2"}]}