PXD025376

PXD025376 is an original dataset announced via ProteomeXchange.

Title	Phosphoproteomics sample preparation impacts biological interpretation of signalling responses
Description	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.
HostingRepository	PRIDE
AnnounceDate	2024-10-07
AnnouncementXML	Submission_2024-10-07_13:39:33.096.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Bharath Sampadi
SpeciesList	scientific name: Mus musculus (Mouse); NCBI TaxID: 10090;
ModificationList	6x(13)C; 6x(13)C; phosphorylated residue; (4,4,5,5-(2)H4)-L-lysine; monohydroxylated residue; 6x(13)C labeled L-arginine; iodoacetamide derivatized residue
Instrument	Q Exactive HF; Q Exactive

Revision	Datetime	Status	ChangeLog Entry
0	2021-04-13 08:43:58	ID requested
⏵ 1	2024-10-07 13:39:33	announced

10.3390/cells10123407;

Sampadi B, Mullenders LHF, Vrieling H, Phosphoproteomics Sample Preparation Impacts Biological Interpretation of Phosphorylation Signaling Outcomes. Cells, 10(12):(2021) [pubmed]

submitter keyword: Phosphoproteomics, TiO2,Mouse, high-throughput phosphoproteomics, SCXPhos, sample preparation, HighPhos, Comparative analysis

Harry Vrieling
contact affiliation	DNA damage responses and cancer lab, Department of Human Genetics, Leiden University Medical Center, Eithovenweg 20, 2333ZC Leiden, The Netherlands.
contact email	h.vrieling@lumc.nl
lab head
Bharath Sampadi
contact affiliation	Leiden University Medical Center
contact email	b.k.sampadi@lumc.nl
dataset submitter

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PRIDE project URI

• PRIDE
  1. PXD025376
     1. Label: PRIDE project
     2. Name: Phosphoproteomics sample preparation impacts biological interpretation of signalling responses