PXD068521-1

PXD068521 is an original dataset announced via ProteomeXchange.

Title	Sequential Denaturation and Protein Precipitation assay (SDPP) improves the sensitivity for ligand target identification at the proteome level
Description	Background: Proteins can be denatured and precipitated under different denaturation conditions, and ligand bound proteins are more resistant to denaturing induced precipitation. Consequently, ligand target proteins can be identified by measuring solubility shifts. Nowadays, numerous methods have been developed to screen ligand target proteins based on different denaturation mechanisms. However, due to the different responses of proteins to different denaturation conditions, there is a significant complementary between these methods. Direct pooling of the denatured supernatants from different denaturation strategies can utilize this complementary, however, the pooling procedure inevitably averages the ligand-induced solubility shift across conditions, significantly compromising target identification sensitivity. Results: To avoid pooling-induced signal compression and enhance identification sensitivity, we developed a novel solubility shift-based approach, termed Sequential Denaturation and Protein Precipitation assay (SDPP). In this strategy, multi-step sequential denaturation of individual sample was conducted, which greatly improves the identification sensitivity. We evaluated the performance of this method by screening the target proteins of Staurosporine, SDPP with thermal treatment followed by organic solvent treatment (TEMP-SL) performs better than other sequential denaturation and one-step denaturation approach, The number of kinase targets identified by SDPP (TEMP-SL) increased by 54%, 38%, 48% and 21%, respectively compared with the TEMP, SL, and pH experiments and IPSSA. We further applied this approach to identify the target proteins of endogenous metabolite cAMP. Except for revealing the known cAMP-PRKAR1A/PRKAR1B interaction, we also observed that the solubility of the C2orf88 responds to the cAMP binding. Significance: This method conducts sequential denaturation on individual samples to induce stepwise protein precipitation, preserving and progressively accumulating the solubility shifts generated at each denaturation step. This cumulative effect amplifies the overall detectable solubility shift signal and greatly improves identification sensitivity, demonstrating great potential as an efficient and high-throughput strategy for proteome-wide ligand target identification.
HostingRepository	jPOST
AnnounceDate	2025-09-29
AnnouncementXML	Submission_2025-09-29_06:11:55.500.xml
DigitalObjectIdentifier
ReviewLevel	Non peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	ynma
SpeciesList	scientific name: Homo sapiens (Human); NCBI TaxID: 9606;
ModificationList	S-carboxamidomethyl-L-cysteine; L-methionine sulfoxide
Instrument	Q Exactive

Revision	Datetime	Status	ChangeLog Entry
0	2025-09-19 00:49:03	ID requested
⏵ 1	2025-09-29 06:11:55	announced

Dataset with its publication pending

submitter keyword: Sequential denaturation

Protein precipitation

Ligand-protein interaction

Drug target screening

Yanni Ma; Mingliang Ye
lab head
ynma
contact affiliation	DICP
dataset submitter

jPOST dataset URI

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