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Phosphoproteomics Sample Preparation Impacts Biological Interpretation of Phosphorylation Signaling Outcomes

The influence of phosphoproteomics sample preparation methods on the biological interpretation of signaling outcome is unclear. Here, we demonstrate a strong bias in phosphorylation signaling targets uncovered by comparing the phosphoproteomes generated by two commonly used methods—strong cation exc...

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Autores principales: Sampadi, Bharath, Mullenders, Leon H. F., Vrieling, Harry
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8699897/
https://www.ncbi.nlm.nih.gov/pubmed/34943915
http://dx.doi.org/10.3390/cells10123407
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author Sampadi, Bharath
Mullenders, Leon H. F.
Vrieling, Harry
author_facet Sampadi, Bharath
Mullenders, Leon H. F.
Vrieling, Harry
author_sort Sampadi, Bharath
collection PubMed
description The influence of phosphoproteomics sample preparation methods on the biological interpretation of signaling outcome is unclear. Here, we demonstrate a strong bias in phosphorylation signaling targets uncovered by comparing the phosphoproteomes generated by two commonly used methods—strong cation exchange chromatography-based phosphoproteomics (SCXPhos) and single-run high-throughput phosphoproteomics (HighPhos). Phosphoproteomes of embryonic stem cells exposed to ionizing radiation (IR) profiled by both methods achieved equivalent coverage (around 20,000 phosphosites), whereas a combined dataset significantly increased the depth (>30,000 phosphosites). While both methods reproducibly quantified a subset of shared IR-responsive phosphosites that represent DNA damage and cell-cycle-related signaling events, most IR-responsive phosphoproteins (>82%) and phosphosites (>96%) were method-specific. Both methods uncovered unique insights into phospho-signaling mediated by single (SCXPhos) versus double/multi-site (HighPhos) phosphorylation events; particularly, each method identified a distinct set of previously unreported IR-responsive kinome/phosphatome (95% disparate) directly impacting the uncovered biology.
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spelling pubmed-86998972021-12-24 Phosphoproteomics Sample Preparation Impacts Biological Interpretation of Phosphorylation Signaling Outcomes Sampadi, Bharath Mullenders, Leon H. F. Vrieling, Harry Cells Article The influence of phosphoproteomics sample preparation methods on the biological interpretation of signaling outcome is unclear. Here, we demonstrate a strong bias in phosphorylation signaling targets uncovered by comparing the phosphoproteomes generated by two commonly used methods—strong cation exchange chromatography-based phosphoproteomics (SCXPhos) and single-run high-throughput phosphoproteomics (HighPhos). Phosphoproteomes of embryonic stem cells exposed to ionizing radiation (IR) profiled by both methods achieved equivalent coverage (around 20,000 phosphosites), whereas a combined dataset significantly increased the depth (>30,000 phosphosites). While both methods reproducibly quantified a subset of shared IR-responsive phosphosites that represent DNA damage and cell-cycle-related signaling events, most IR-responsive phosphoproteins (>82%) and phosphosites (>96%) were method-specific. Both methods uncovered unique insights into phospho-signaling mediated by single (SCXPhos) versus double/multi-site (HighPhos) phosphorylation events; particularly, each method identified a distinct set of previously unreported IR-responsive kinome/phosphatome (95% disparate) directly impacting the uncovered biology. MDPI 2021-12-03 /pmc/articles/PMC8699897/ /pubmed/34943915 http://dx.doi.org/10.3390/cells10123407 Text en © 2021 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Sampadi, Bharath
Mullenders, Leon H. F.
Vrieling, Harry
Phosphoproteomics Sample Preparation Impacts Biological Interpretation of Phosphorylation Signaling Outcomes
title Phosphoproteomics Sample Preparation Impacts Biological Interpretation of Phosphorylation Signaling Outcomes
title_full Phosphoproteomics Sample Preparation Impacts Biological Interpretation of Phosphorylation Signaling Outcomes
title_fullStr Phosphoproteomics Sample Preparation Impacts Biological Interpretation of Phosphorylation Signaling Outcomes
title_full_unstemmed Phosphoproteomics Sample Preparation Impacts Biological Interpretation of Phosphorylation Signaling Outcomes
title_short Phosphoproteomics Sample Preparation Impacts Biological Interpretation of Phosphorylation Signaling Outcomes
title_sort phosphoproteomics sample preparation impacts biological interpretation of phosphorylation signaling outcomes
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8699897/
https://www.ncbi.nlm.nih.gov/pubmed/34943915
http://dx.doi.org/10.3390/cells10123407
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