Phosphoproteomics Sample Preparation Impacts Biological Interpretation of Phosphorylation Signaling Outcomes
Cells, vol. 10, pp. 3407
Abstract
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.
Authors 3
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Leiden University Medical Center
Affiliation as printed
Department of Human Genetics, Leiden University Medical Center, Einthovenweg 20, 2333 ZC Leiden, The Netherlands
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Nagoya University · Leiden University Medical Center
Affiliation as printed
Department of Genetics, Research Institute of Environmental Medicine (RIeM), Nagoya University, Nagoya 464-0805, Aichi, Japan
Department of Human Genetics, Leiden University Medical Center, Einthovenweg 20, 2333 ZC Leiden, The Netherlands
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Leiden University Medical Center
Affiliation as printed
Department of Human Genetics, Leiden University Medical Center, Einthovenweg 20, 2333 ZC Leiden, The Netherlands
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