PXD059986

PXD059986 is an original dataset announced via ProteomeXchange.

Title	Development and Application of a Targeted Phosphoproteomics Method for mTOR Pathway Analysis in Zebrafish PAC2 Cells
Description	The mechanistic target of rapamycin (mTOR) signaling pathway plays a crucial role in regulating cellular growth and proliferation. While extensively studied in mammals, the phosphorylation dynamics of this pathway in non-mammalian model organisms remain largely unexplored, partially because of the scarcity of suitable antibodies to measure (phosphorylated) proteins of interest. To address this challenge, we developed a novel, antibody-independent targeted phosphoproteomics approach for quantifying mTOR pathway protein abundance and phosphorylation levels in zebrafish (Danio rerio) using liquid chromatography-tandem mass spectrometry (LC-MS/MS). With optimized sample processing and data analysis workflows, our method demonstrates excellent linearity (R2 > 0.97) over two orders of magnitude, with limits of quantification as low as 1.2 fmol·µL-1 for phosphopeptides. It further addresses key analytical challenges in phosphoproteomics, including efficient enrichment, accurate quantification of low-abundance phosphopeptides, and correction for sample loss during sample preparation procedures. Lastly, our approach allows for a simultaneous analysis of both phosphorylated and non-phosphorylated peptides, thus facilitating the differentiation between abundance-driven changes and true (de)phosphorylation events. Using our method, we successfully quantified 10 endogenous phosphosites and 15 endogenous proteins in zebrafish PAC2 cells at different cell culture growth phases, revealing complex phosphorylation dynamics within the mTOR pathway. This work demonstrates the high potential of the LC-MS/MS-based analytical approaches for investigating phosphorylation-governed signalling dynamics in non-mammalian models, thus paving the way for developing valuable tools for comparative studies, toxicological investigations, and exploration of phosphorylation dynamics across species.
HostingRepository	PanoramaPublic
AnnounceDate	2025-01-27
AnnouncementXML	Submission_2025-01-27_09:07:40.144.xml
DigitalObjectIdentifier
ReviewLevel	Non peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Supported dataset by repository
PrimarySubmitter	Nikolai Huwa
SpeciesList	scientific name: Danio rerio; NCBI TaxID: 7955;
ModificationList	Phospho; Carbamidomethyl; Label:13C(6)15N(2); Label:13C(6)15N(4); Label:13C(9)15N(1); Label:13C(5)15N(1)
Instrument	6495C Triple Quadrupole LC/MS

Revision	Datetime	Status	ChangeLog Entry
0	2025-01-20 10:25:38	ID requested
⏵ 1	2025-01-27 09:07:40	announced

no publication

submitter keyword: Phosphoproteomics mTOR pathway Zebrafish LC-MS/MS Targeted proteomics Growth phase dynamics NTA-Fe3+ enrichment

Ksenia Groh
contact affiliation	Eawag, Swiss Federal Institute of Aquatic Science and Technology, 8600 Dübendorf, Switzerland
contact email	ksenia.groh@eawag.ch
lab head
Nikolai Huwa
contact affiliation	Eawag, Swiss Federal Institute of Aquatic Science and Technology, 8600 Dübendorf, Switzerland
contact email	nikolai.huwa@eawag.ch
dataset submitter

Panorama Public dataset URI