Single-Shot Capillary Zone Electrophoresis-Tandem Mass Spectrometry Produces over 4400 Phosphopeptide Identifications from a 220 ng Sample.

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In this work, we coupled CZE to an Orbitrap Fusion Lumos Tribrid platform with an advanced peak determination algorithm for phosphoproteomics analysis. A linear-polyacrylamide-coated capillary with very low electroosmotic flow was used for the separation. The optimal injection volume was between 100 and 150 nL of a solution of phosphopeptides in 30 mM ammonium bicarbonate (pH 8.2) buffer, which produces a dynamic pH junction sample injection. Larger injection volumes resulted in serious peak broadening and decreased numbers of phosphopeptide identifications. The optimized system identified 4405 phosphopeptides from 220 ng of enriched phosphopeptides from mouse brain, which represents the state-of-the-art result for single-shot CZE-ESI-MS/MS-based phosphoproteome analysis. We found that the migration time for phosphopeptides is much longer than that for non-phosphopeptides and increased along with the number of phosphorylation sites on the peptides, as expected for the additional negative charges associated with the phosphate groups. We also investigated the phosphorylation site motifs; a number of motifs appeared in the CZE-ESI-MS/MS data but not in LC-ESI-MS/MS data, which suggested the complementary performance of the techniques. The data are available via ProteomeXchange with identifier PXD012888.", "claims": [{"id": "claim-method", "statement": "Scatter plot between replicates was shown in Figure 3, pearson correlation coefficient of 0.974 is obtained, demonstrating the good reproducibility and high label-free quantification precision of the CZE-MS method.", "kind": "methodological", "target": null, "evidence": [{"asset": "source:pmid:31180221", "locator": "sections[11] \"Coupling to an Orbitrap Fusion Lumos Tribrid platform with APD algorithm.\" paragraph 2", "quote": "Scatter plot between replicates was shown in Figure 3, pearson correlation coefficient of 0.974 is obtained, demonstrating the good reproducibility and high label-free quantification precision of the CZE-MS method."}], "state": "complete"}, {"id": "claim-data-access", "statement": "Data are available via ProteomeXchange with identifier PXD012888.", "kind": "methodological", "target": null, "evidence": [{"asset": "source:pmid:31180221", "locator": "sections[0] \"Abstract\" paragraph 1", "quote": "Data are available via ProteomeXchange with identifier PXD012888."}], "state": "complete"}, {"id": "claim-target-result", "statement": "The optimized system identified 4405 phosphopeptides from 220 ng of enriched phosphopeptides from mouse brain.", "kind": "qualitative", "target": null, "evidence": [{"asset": "source:pmid:31180221", "locator": "sections[0] \"Abstract\" paragraph 1", "quote": "The optimized system identified 4405 phosphopeptides from 220 ng of enriched phosphopeptides from mouse brain, which represents the state-of-the-art result for single shot CZE-ESI-MS/MS based phosphoproteome analysis."}], "state": "complete"}], "gaps": []}, "experiment": {"state": "complete", "objective": "The optimized system identified 4405 phosphopeptides from 220 ng of enriched phosphopeptides from mouse brain.", "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:31180221"], "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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