PXD014565

PXD014565 is an original dataset announced via ProteomeXchange.

Title	A systematic comparison of label-free, SILAC and TMT techniques to study early adaption towards inhibition of EGFR signaling in the colorectal cancer cell line Difi
Description	The epithelial growth factor receptor (EGFR) signalling network plays an essential role inproliferation and survival of colorectal cancer cells. For this reason, EGFR is targeted using therapeutic antibodies blocking EGFR signalling (e.g. cetuximab). However, anti-EGFR therapy is rarely curative as subpopulations of tumor cells survive the initial treatment and develop therapy-resistance. To analyse cellular signalling and assess the quantitative changes of the proteome and the phosphoproteome several proteomics approaches have been established. Here, we compared the widely used quantification strategies label-free (LF), SILAC and TMT and systematically evaluated their technical characteristics including coverage, technical variability, complementarity and the ability to assess statistical significant differences. We studied the dynamics of the EGFR signalling network upon treatment withcetuximab after 0 h, 3 h and 24 h, representing the first cellular adaption towards anti-EGFR therapy, with the aim to identify the most powerful approach for monitoring cellular signalling in this setup. We demonstrate that LF had a superior coverage regarding quantification of both, the proteome and phosphoproteome, while SILAC exhibited the highest precision resulting in superior assessment of differentially abundant proteins and class I phosphosites (p-sites). TMT was outperformed by the other two approaches, illustrating its critical dependency on the applied labelling-design. On the protein level, we observed only little regulation upon cetuximab treatment, whereas a great fraction of p-sites was significantly regulated after 3 h and 24 h. The dynamics of significantly regulated p-sites illustrated an initial downregulation of the EGFR and MAPK signalling pathways, which was partially rescued as soon as after 24h. We identified upregulation and signalling via ERBB3 as a possible mechanism bypassing the blockage of EGFR. Moreover, phosphorylation motifs associated to calcium signalling were continuously upregulated, thereby representing compensatory signalling presumably resulting in the observed reactivation of MAPK1/3 (Erk1/2).
HostingRepository	PRIDE
AnnounceDate	2019-12-11
AnnouncementXML	Submission_2019-12-11_04:08:17.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Markus Stepath
SpeciesList	scientific name: Homo sapiens (Human); NCBI TaxID: 9606;
ModificationList	phosphorylated residue; monohydroxylated residue; iodoacetamide derivatized residue
Instrument	Q Exactive

Revision	Datetime	Status	ChangeLog Entry
0	2019-07-11 01:01:29	ID requested
⏵ 1	2019-12-11 04:08:19	announced

Stepath M, Z, ü, lch B, Maghnouj A, Schork K, Turewicz M, Eisenacher M, Hahn S, Sitek B, Bracht T, Systematic Comparison of Label-Free, SILAC, and TMT Techniques to Study Early Adaption toward Inhibition of EGFR Signaling in the Colorectal Cancer Cell Line DiFi. J Proteome Res, 19(2):926-937(2020) [pubmed]

curator keyword: Technical, Biomedical

submitter keyword: Colorectal cancer, SILAC, TMT, label-free, Cetuximab, anti-EGFR therapy, ERBB3, MAPK, Phosphoproteomics, Adaptive mechanism

Barbara Sitek
contact affiliation	Medizinisches Proteom-Center, Ruhr-Universität Bochum, Bochum, Germany
contact email	barbara.sitek@rub.de
lab head
Markus Stepath
contact affiliation	Ruhr University Bochum - Medizinisches Proteom-Center
contact email	markus.stepath@rub.de
dataset submitter

Dataset FTP location NOTE: Most web browsers have now discontinued native support for FTP access within the browser window. But you can usually install another FTP app (we recommend FileZilla) and configure your browser to launch the external application when you click on this FTP link. Or otherwise, launch an app that supports FTP (like FileZilla) and use this address: ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2019/12/PXD014565

PRIDE project URI

• PRIDE
  1. PXD014565
     1. Label: PRIDE project
     2. Name: A systematic comparison of label-free, SILAC and TMT techniques to study early adaption towards inhibition of EGFR signaling in the colorectal cancer cell line Difi