Accurate label-free protein quantitation with high- and low-resolution mass spectrometers.

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We directly compared MS1 and MS2 quantitation by analyzing human protein standards diluted into Escherichia coli extracts on an Orbitrap mass spectrometer. We found that summed MS2 intensities were nearly as accurate as integrated MS1 intensities, and both outperformed MS2 spectral counting in accuracy and linearity. We compared these results to those obtained from two low-resolution ion-trap mass spectrometers; summed MS2 intensities from LTQ and LTQ Velos instruments were similar in accuracy to those from the Orbitrap. Data from all three instruments are available via ProteomeXchange with identifier PXD000602. Abundance measurements using MS1 or MS2 intensities had limitations, however. While measured protein concentration was on average well-correlated with the known concentration, there was considerable protein-to-protein variation. Moreover, not all human proteins diluted to a mole fraction of 10(-3) or lower were detected, with a strong falloff below 10(-4) mole fraction. These results show that MS1 and MS2 intensities are simple measures of protein abundance that are on average accurate but should be limited to quantitation of proteins of intermediate to higher fractional abundance.", "claims": [{"id": "claim-target-result", "statement": "Summed MS2 intensities were nearly as accurate as integrated MS1 intensities, and both outperformed MS2 spectral counting in accuracy and linearity.", "kind": "qualitative", "target": null, "evidence": [{"asset": "source:pmid:24295401", "locator": "sections[0] \"Abstract\" paragraph 1", "quote": "We found that summed MS2 intensities were nearly as accurate as integrated MS1 intensities, and both outperformed MS2 spectral counting in accuracy and linearity."}, {"asset": "source:pmid:24295401", "locator": "sections[11] \"Relationship between mole fraction and MS1 quantitation\" paragraph 4", "quote": "The fits for the relationship between known mole fraction and riBAQ or rTop3 were not significantly different from the unity line on a log-log plot (Fig. 3A,B)."}, {"asset": "source:pmid:24295401", "locator": "sections[0] \"Abstract\" paragraph 1", "quote": "Data from all three instruments are available via ProteomeXchange with identifier PXD000602."}], "state": "complete"}], "gaps": []}, "experiment": {"state": "partial", "objective": "Summed MS2 intensities were nearly as accurate as integrated MS1 intensities, and both outperformed MS2 spectral counting in accuracy and linearity.", "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:24295401"], "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. 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For each mass spectrometer, 24 different LC-MS/MS runs were conducted: four separate experimen", "and each generating six peptide samples for LC-MS/MS. Supplementary Material 1_si_001 2_si_003 3_", "r the UPS1 experiments, the entire Universal Proteomics Standard Set (Sigma-Aldrich) was diluted int"], "order_basis": "paper_text_order:102", "id": "assay:1"}]}