PXD059986

PXD059986 is an original dataset announced via ProteomeXchange.

Title	Development and Application of a Targeted Phosphoproteomics Method for Analysing the mTOR Pathway Dynamics in Zebrafish PAC2 Cell Line
Description	The mechanistic target of rapamycin (mTOR) signalling 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, often due to the scarcity of suitable antibodies to measure (phosphorylated) proteins of interest. To address this gap, we developed an antibody-independent targeted phosphoproteomics method applying liquid chromatography-tandem mass spectrometry (LC-MS/MS) to quantify the abundance and phosphorylation levels of mTOR pathway-related proteins in zebrafish (Danio rerio), using the permanent cell line PAC2 as a model system. With optimized sample processing and data analysis strategies, we could successfully quantify 10 endogenous phosphosites and 15 endogenous proteins at different cell culture growth phases, revealing complex phosphorylation dynamics for both the upstream regulators (e.g., AKT, AMPK) and downstream effectors (e.g., eIF4EBP1, RPS6) of the mTOR pathway, which reflected transition from exponential growth to stationary subsistence. Our findings confirm the overall similarity of the mTOR pathway structure and functionality between zebrafish and mammals. Furthermore, this work demonstrates the high potential of the LC-MS/MS-based analytical approaches for studying phosphorylation-governed signalling in diverse organisms of interest, thus paving the way for further investigations in comparative physiology and toxicology across species.
HostingRepository	PanoramaPublic
AnnounceDate	2025-08-13
AnnouncementXML	Submission_2025-08-13_11:42:00.725.xml
DigitalObjectIdentifier
ReviewLevel	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
⏵ 2	2025-08-13 11:42:01	announced	: Added publication

Huwa, N., Schönenberger, R., & Groh, K. J. (2025). Development and application of a targeted phosphoproteomics method for analysing the mTOR pathway dynamics in zebrafish PAC2 cell line. Journal of Proteomics, 319, 105469.

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