Anion-exchange chromatography of phosphopeptides: weak anion exchange versus strong anion exchange and anion-exchange chromatography versus electrostatic repulsion-hydrophilic interaction chromatography.

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This study compares the potential and limitations of electrostatic repulsion-hydrophilic interaction chromatography (ERLIC) and anion-exchange chromatography (AEX). At a pH higher than 5, phosphopeptides have two negative charges per residue and are well-retained in AEX. However, peptides with one or two phosphate groups are not separated from peptides with multiple Asp or Glu residues, interfering with the identification of phosphopeptides. At a pH of 2, phosphate residues have just a single negative charge but Asp and Glu are uncharged. This facilitates the separation of phosphopeptides from unmodified acidic peptides. Singly phosphorylated peptides are retained weakly under these conditions, due to electrostatic repulsion, unless hydrophilic interaction is superimposed in the ERLIC mode. Weak anion-exchange (WAX) and strong anion-exchange (SAX) columns were compared, with both peptide standards and a HeLa cell tryptic digest. The SAX column exhibited greater retention at pH 6 than did the WAX column. However, only about 60% as many phosphopeptides were identified with SAX at pH 6 than via ERLIC at pH 2. In one ERLIC run, 12 467 phosphopeptides were identified, including 4233 with more than one phosphate. We conclude that chromatography of phosphopeptides is best performed at low pH in the ERLIC mode. Under those conditions, the performances of the SAX and WAX materials were comparable. The data have been deposited with the ProteomeXchange with identifier PXD001333.", "claims": [{"id": "claim-method", "statement": "The result was filtered to 1% false discovery rate using the Percolator algorithm26 integrated in Proteome Discoverer.", "kind": "methodological", "target": null, "evidence": [{"asset": "source:pmid:25827581", "locator": "sections[12] \"Data Analysis\" paragraph 2", "quote": "The result was filtered to 1% false discovery rate using the Percolator algorithm26 integrated in Proteome Discoverer."}], "state": "complete"}, {"id": "claim-data-access", "statement": "The mass spectrometry proteomics data in this paper have been deposited with the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE partner repository37 with the support of PRIDE team EBI, with data set identifier PXD001333.", "kind": "methodological", "target": null, "evidence": [{"asset": "source:pmid:25827581", "locator": "sections[20] \"Accession Codes\" paragraph 2", "quote": "The mass spectrometry proteomics data in this paper have been deposited with the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE partner repository37 with the support of PRIDE team EBI, with data set identifier PXD001333."}], "state": "complete"}, {"id": "claim-target-result", "statement": "Chromatography of phosphopeptides is best performed at low pH in the ERLIC mode, which identified more phosphopeptides from a HeLa digest than high-pH AEX.", "kind": "qualitative", "target": null, "evidence": [{"asset": "source:pmid:25827581", "locator": "sections[0] \"Abstract\" paragraph 1", "quote": "We conclude that chromatography of phosphopeptides is best performed at low pH in the ERLIC mode."}, {"asset": "source:pmid:25827581", "locator": "sections[0] \"Abstract\" paragraph 1", "quote": "In one ERLIC run, 12 467 phosphopeptides were identified, including 4233 with more than one phosphate."}, {"asset": "source:pmid:25827581", "locator": "sections[0] \"Abstract\" paragraph 1", "quote": "The data have been deposited with the ProteomeXchange with identifier PXD001333."}], "state": "complete"}], "gaps": []}, "experiment": {"state": "complete", "objective": "Chromatography of phosphopeptides is best performed at low pH in the ERLIC mode, which identified more phosphopeptides from a HeLa digest than high-pH AEX.", "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:25827581"], "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. 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