PXD047646

PXD047646 is an original dataset announced via ProteomeXchange.

Title	A Rapid One-pot Workflow for Sensitive Microscale Phosphoproteomics
Description	Rapid advancement of single-cell proteomics has revolutionized modern research to reveal cell-specific regulation and cellular phenotypes. Though phosphoproteomics is a powerful method for mapping signal transduction networks, its sensitivity has lagged behind due to significantly lower abundance, complex sample preparation and substantial sample input. Here, we present a simple and rapid one-pot phosphoproteomics workflow (SOP-Phos) integrated with data-independent acquisition mass spectrometry (DIA MS) for microscale phosphoproteomic analysis. SOP-Phos adapts sodium deoxycholate based one-step lysis, reduction and alkylation, direct trypsinization and phosphopeptide enrichment by TiO2 beads in a single-tube format. By reducing surface adsorptive losses via utilizing n-Dodecyl β-D-maltoside pre-coated tubes throughout the workflow, and shortening digestion time, SOP-Phos is completed within 3-4-hr with 1.4-fold higher identification coverage, especially on recovering longer and multiple phosphopeptides. SOP-Phos coupled with DIA demonstrated >90% specificity, enhanced sensitivity, lower missing values (<1%), and improved quantitative reproducibility (8%-10% CV). We established a sample size-comparable spectral library to enhance 2.6-6.4-fold more phosphopeptides compared to directDIA, offering coverage of 33,787±670 to 22,070±861 phosphopeptides from 5 to 0.5 µg (~2500 cells) cell lysate. Such sensitivity enabled mapping key lung cancer signaling sites such as autophosphorylation sites Y1197/Y1172 of EGFR and drug targets. The feasibility of SOP-Phos-DIA was demonstrated on two pairs of EGFR-Tyrosine Kinase Inhibitor (TKI)- sensitive and resistant cells, revealing the interplay of multi-pathway Hippo-EGFR-ERBB signaling cascades which not only provides the mechanistic insight to EGFR-TKI resistance but also suggest combination therapy to restore the sensitivity to EGFR-TKI therapy. Using the commonly used reagents, the SOP-Phos-DIA is an efficient and robust protocol that can easily be adapted in the community for microscale phosphoproteomic analysis with deep depth to decipher biology from low-input samples such as primary or rare immune cells.
HostingRepository	jPOST
AnnounceDate	2025-05-06
AnnouncementXML	Submission_2025-05-05_08:00:05.114.xml
DigitalObjectIdentifier
ReviewLevel	Non peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Gul Muneer
SpeciesList	scientific name: Homo sapiens (Human); NCBI TaxID: 9606;
ModificationList	S-carboxamidomethyl-L-cysteine; alpha-amino acetylated residue; L-methionine sulfoxide; O-phospho-L-serine; O-phospho-L-threonine; O4'-phospho-L-tyrosine
Instrument	LTQ Orbitrap

Revision	Datetime	Status	ChangeLog Entry
0	2023-12-08 04:12:59	ID requested
⏵ 1	2025-05-05 08:00:05	announced

Dataset with its publication pending

submitter keyword: Phosphoproteomics

Data-independent Acquisition

Sample Preparation

Lung Cancer

EGFR-Tyrosine Kinase Inhibitor (TKI)

Yu-Ju Chen
lab head
Gul Muneer
contact affiliation	Institute of Chemistry, Academia Sinica, Taipei, Taiwan
dataset submitter

jPOST dataset URI

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