dc.contributor.author | Bortel, Patricia | |
dc.contributor.author | Piga, Ilaria | |
dc.contributor.author | Koenig, Claire | |
dc.contributor.author | Gerner, Christopher | |
dc.contributor.author | Martinez-Val, Ana | |
dc.contributor.author | Olsen, Jesper V | |
dc.date.accessioned | 2024-07-04T12:27:29Z | |
dc.date.available | 2024-07-04T12:27:29Z | |
dc.date.issued | 2024-05 | |
dc.identifier.citation | Mol Cell Proteomics. 2024 May;23(5):100754. | es_ES |
dc.identifier.uri | http://hdl.handle.net/20.500.12105/20077 | |
dc.description.abstract | Improving coverage, robustness, and sensitivity is crucial for routine phosphoproteomics analysis by single-shot liquid chromatography-tandem mass spectrometry (LC-MS/MS) from minimal peptide inputs. Here, we systematically optimized key experimental parameters for automated on-bead phosphoproteomics sample preparation with a focus on low-input samples. Assessing the number of identified phosphopeptides, enrichment efficiency, site localization scores, and relative enrichment of multiply-phosphorylated peptides pinpointed critical variables influencing the resulting phosphoproteome. Optimizing glycolic acid concentration in the loading buffer, percentage of ammonium hydroxide in the elution buffer, peptide-to-beads ratio, binding time, sample, and loading buffer volumes allowed us to confidently identify >16,000 phosphopeptides in half-an-hour LC-MS/MS on an Orbitrap Exploris 480 using 30 μg of peptides as starting material. Furthermore, we evaluated how sequential enrichment can boost phosphoproteome coverage and showed that pooling fractions into a single LC-MS/MS analysis increased the depth. We also present an alternative phosphopeptide enrichment strategy based on stepwise addition of beads thereby boosting phosphoproteome coverage by 20%. Finally, we applied our optimized strategy to evaluate phosphoproteome depth with the Orbitrap Astral MS using a cell dilution series and were able to identify >32,000 phosphopeptides from 0.5 million HeLa cells in half-an-hour LC-MS/MS using narrow-window data-independent acquisition (nDIA). | es_ES |
dc.description.sponsorship | Funding and additional information—Work at The Novo
Nordisk Foundation Center for Protein Research (CPR) is
funded in part by a donation from the Novo Nordisk Foundation (NNF14CC0001). This work has also been funded as
part of EPIC-XS project under the grant agreement no. 823839
funded by the Horizon 2020 programme of the European
Union. P.B. was supported by the “International Exchange
Program” (Vienna Doctoral School in Chemistry, DoSChem),
the funding program “Internationale Kommunikation” (Österreichische Forschungsgemeinschaft, ÖFG) and the “Erasmus+ Traineeship Mobility” program. C.K. is supported by the
Marie Skłodowska Curie European Training Network
“PUSHH” (grant number No. 861389). | es_ES |
dc.language.iso | eng | es_ES |
dc.publisher | Elsevier | es_ES |
dc.type.hasVersion | VoR | es_ES |
dc.rights.uri | http://creativecommons.org/licenses/by/4.0/ | * |
dc.subject.mesh | Phosphopeptides | es_ES |
dc.subject.mesh | Proteomics | es_ES |
dc.subject.mesh | Tandem Mass Spectrometry | es_ES |
dc.subject.mesh | Phosphoproteins | es_ES |
dc.subject.mesh | Humans | es_ES |
dc.subject.mesh | Chromatography, Liquid | es_ES |
dc.subject.mesh | HeLa Cells | es_ES |
dc.subject.mesh | Proteome | es_ES |
dc.subject.mesh | Phosphorylation | es_ES |
dc.subject.mesh | Automation | es_ES |
dc.title | Systematic Optimization of Automated Phosphopeptide Enrichment for High-Sensitivity Phosphoproteomics. | es_ES |
dc.type | journal article | es_ES |
dc.rights.license | Atribución 4.0 Internacional | * |
dc.identifier.pubmedID | 38548019 | es_ES |
dc.format.volume | 23 | es_ES |
dc.format.number | 5 | es_ES |
dc.format.page | 100754 | es_ES |
dc.identifier.doi | 10.1016/j.mcpro.2024.100754 | es_ES |
dc.contributor.funder | Novo Nordisk Foundation | es_ES |
dc.contributor.funder | Unión Europea. Comisión Europea. H2020 | es_ES |
dc.contributor.funder | Marie Curie | es_ES |
dc.description.peerreviewed | Sí | es_ES |
dc.identifier.e-issn | 1535-9484 | es_ES |
dc.relation.publisherversion | 10.1016/j.mcpro.2024.100754 | es_ES |
dc.identifier.journal | Molecular & cellular proteomics : MCP | es_ES |
dc.repisalud.orgCNIC | CNIC::Grupos de investigación::Proteómica cardiovascular | es_ES |
dc.repisalud.institucion | CNIC | es_ES |
dc.relation.projectID | info:eu-repo/grantAgreement/EC/H2020/861389 | es_ES |
dc.rights.accessRights | open access | es_ES |