<<< Full experiment listing

PXD002363

PXD002363 is an original dataset announced via ProteomeXchange.

Dataset Summary
TitlePhosphoproteomic profiling of the DNA damage response in MCF10A and human PBMC cells by SILAC labeling and 1D and 2D-LC-MS/MS
DescriptionThe DNA-damage response (DDR) is a critical network for maintaining genomic integrity, and mutations in the DDR are among the most frequently identified in tumors. Phosphorylation is a key signaling event in the DDR network. We sought to design high throughput, mass spectrometry based assays to monitor phosphopeptides in the DDR network for biological experiments and pharmacodynamics studies. These assays require an enrichment step, which can be done using antibodies. Developing antibodies de novo for phosphopeptide enrichment is costly and time-consuming, so we assessed the feasibility of immobilized-metal affinity chromatography (IMAC) enrichment coupled with multiple reaction monitoring-mass spectrometry (MRM-MS) for precise measurement of large numbers of phosphopeptides in the DDR network. Towards this goal, we evaluate the ability of the approach to reproducibly measure the endogenous phosphorylation levels of proteins associated with the DDR. Reproducibly detectable phosphopeptide targets for MRM-based assay development were empirically identified from LC-MS/MS analyses of SILAC-labeled MCF10A human breast epithelial cells exposed or mock-exposed to DNA damage. Samples were treated in triplicate with either ionizing radiation (IR) or methyl methanesulfonate (MMS) as the DNA-damaging agent. SILAC-labeled cells were prepared in two separate (label swap) experiments. First, treated cell lines were grown in heavy SILAC medium and untreated in light, and second, this labeling scheme was reversed. Untreated and treated cells were mixed at a 1:1 ratio by protein mass. To identify phosphopeptides at levels observable by MRM in an approach amenable to moderate throughput, a single step IMAC enrichment was developed for rapid processing of whole cell lysate.
HostingRepositoryPRIDE
AnnounceDate2015-12-01
AnnouncementXMLSubmission_2015-12-02_05:44:15.xml
DigitalObjectIdentifier
ReviewLevelPeer-reviewed dataset
DatasetOriginOriginal dataset
RepositorySupportUnsupported dataset by repository
PrimarySubmitterJacob Kennedy
SpeciesList scientific name: Homo sapiens (Human); NCBI TaxID: 9606;
ModificationListphosphorylated residue; carbamoylated residue
InstrumentLTQ Orbitrap Velos; LTQ Orbitrap Elite
Dataset History
RevisionDatetimeStatusChangeLog Entry
02015-06-12 00:51:45ID requested
12015-12-01 15:28:18announced
22015-12-02 05:44:16announcedUpdated publication reference for PubMed record(s): 26621847.
Publication List
Kennedy JJ, Yan P, Zhao L, Ivey RG, Voytovich UJ, Moore HD, Lin C, Pogosova-Agadjanyan EL, Stirewalt DL, Reding KW, Whiteaker JR, Paulovich AG, Immobilized Metal Affinity Chromatography Coupled to Multiple Reaction Monitoring Enables Reproducible Quantification of Phospho-signaling. Mol Cell Proteomics, 15(2):726-39(2016) [pubmed]
Keyword List
curator keyword: Biomedical, Biological
submitter keyword: Human, MCF10A, PBMC, IR, MMS, SILAC, LC-MS/MS
Contact List
Amanda G Paulovich
contact affiliationFred Hutchinson Cancer Research Center
contact emailapaulovi@fredhutch.org
lab head
Jacob Kennedy
contact affiliationClinical Research Division
contact emailjkennedy@fhcrc.org
dataset submitter
Full Dataset Link List
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/2015/12/PXD002363
PRIDE project URI
Repository Record List
[ + ]