Dataset Information

Phosphoproteomics sample preparation impacts biological interpretation of signalling responses

ABSTRACT: Quantitative data-dependent acquisition phosphoproteomics (DDAP) has revolutionised cell signaling studies. However, the impact of the specific DDAP sample preparation method on the phosphoproteome quantified is unclear as each study quantifies only a fraction of the phosphoproteome. Here, we compared two distinct phosphoproteome datasets profiled either by a simple or a complex workflow. The complex workflow involved a strong-cation exchange chromatography-based phosphoproteomics (SCXPhos) and the simple workflow involved a modified single-run high-throughput EasyPhos-based phosphoproteomics (HighPhos). We used mouse embryonic stem cells as a biological system for the comparison and profiled phosphoproteomes at two different time points after exposure to two different doses of ionizing radiation (IR). Both methods achieved equal coverage of ~20,000 phosphosites in total, whereas combined profiling resulted in a dramatic increase in the depth of the phosphoproteome (>30,000 phosphosites). Over 96% of the identified IR-responsive phosphosites were method-specific. Despite such limited overlap, both methods uncovered a prominent role for ATM-mediated phosphorylation and reproducibly quantified a subset of shared IR-responsive phosphosites. The majority of IR-responsive phosphosites identified by SCXPhos concerned single phosphorylation events, whereas double- and multi-site phosphorylation events predominated after HighPhos. The coverage of the phosphoproteome increased to >82,000 phosphosites when multiple datasets were included suggesting that current global phosphoproteome studies are nowhere near representing the entire spectrum of phosphorylation events. Thus, our results demonstrate the power of the combination of different methodologies to obtain a better representative image of a broad view of phosphorylation signaling events.

INSTRUMENT(S): Q Exactive HF, Q Exactive

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Cell Culture, Embryonic Stem Cell

SUBMITTER: Bharath Sampadi

LAB HEAD: Harry Vrieling

PROVIDER: PXD025376 | Pride | 2021-12-25

REPOSITORIES: Pride

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			Action	DRS
	20150313_QE_q101170a_BSamp_J1_0.5h_SCX_TiO2_F1_Bio1.raw	Raw
	20150313_QE_q101171a_BSamp_J1_0.5h_SCX_TiO2_F2_Bio1.raw	Raw
	20150313_QE_q101172a_BSamp_J1_0.5h_SCX_TiO2_F3_Bio1.raw	Raw
	20150313_QE_q101173a_BSamp_J1_0.5h_SCX_TiO2_F4_Bio1.raw	Raw
	20150313_QE_q101174a_BSamp_J1_0.5h_SCX_TiO2_F5_Bio1.raw	Raw

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Phosphoproteomics Sample Preparation Impacts Biological Interpretation of Phosphorylation Signaling Outcomes.

Sampadi Bharath B Mullenders Leon H F LHF Vrieling Harry H

Cells 20211203 12

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 ...[more]

PMID: 34943915

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