PXD017571

PXD017571 is an original dataset announced via ProteomeXchange.

Title	An Approach to Spatiotemporally Resolve Protein Interaction Networks in Living Cells - B2AR_localization
Description	SRM assays were generated for selected interactors of B2AR and DOR (MSV000084857), as well as for localization controls and ribosomal proteins (RPL18A, RPL28, RPL3, RPL35A, RPL6) as internal controls for normalization (Table S5 in publication). We selected localization markers for different cellular compartments and monitored them across the time points for B2AR and the spatial references (ENDO, CYTO, PM). The main goal of this experiment was to demonstrate that we can use the proximity labeling data to determine the localization of the receptor over the time course after receptor activation. SRM assay generation was performed using Skyline. For all targeted proteins, proteotypic peptides and optimal transitions for identification and quantification were selected based on a spectral library generated from the shotgun MS experiments. The Skyline spectral library was used to extract optimal coordinates for the SRM assays, e.g., peptide fragments and peptide retention times. For each protein 1-4 peptides were selected based on intensity, peptide length as well as chromatographic performance. For each peptide the 4 best SRM transitions were selected based on intensity and peak shape. Digested peptide mixtures were analyzed by LC-SRM on a Thermo Scientific TSQ Quantiva MS system equipped with a Proxeon Easy nLC 1200 ultra high-pressure liquid chromatography and autosampler system. Samples were injected onto a C18 column (25 cm x 75 mm I.D. packed with ReproSil Pur C18 AQ 1.9 mm particles) in 0.1% formic acid and then separated with an 80 min gradient from 5% to 40% Buffer B (90% ACN/10% water/0.1% formic acid) at a flow rate of 300 nl/min. SRM acquisition was performed operating Q1 and Q3 at 0.7 unit mass resolution. For each peptide the best 4 transitions were monitored in a scheduled fashion with a retention time window of 4 min and a cycle time fixed to 2s. Argon was used as the collision gas at a nominal pressure of 1.5 mTorr. Collision energies were calculated by, CE = 0.0348 * (m/z) + 0.4551 and CE = 0.0271 * (m/z) + 1.5910 (CE, collision energy and m/z, mass to charge ratio) for doubly and triply charged precursor ions, respectively. RF lens voltages were calculated by, RF= 0.1088 * (m/z) + 21.029 and RF= 0.1157 * (m/z) + 0.1157 (RF, RF lens voltage and m/z, mass to charge ratio) for doubly and triply charged precursor ions, respectively. SRM data were processed using Skyline. Protein significance analysis was performed using MSstats. Normalization across samples was conducted based on selected global standard proteins (RPL18A, RPL28, RPL3, RPL35A, RPL6). Each protein was tested for abundance differences comparing DOR-APEX2 time points to the spatial references, PM-APEX2 and ENDO-APEX2. Proteins with an adjusted p-value < 0.05 were considered significant.
HostingRepository	MassIVE
AnnounceDate	2020-02-17
AnnouncementXML	Submission_2020-02-17_19:01:47.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Meena Choi
SpeciesList	scientific name: Homo sapiens; common name: human; NCBI TaxID: 9606;
ModificationList	No PTMs are included in the dataset
Instrument	TSQ Quantiva

Revision	Datetime	Status	ChangeLog Entry
0	2020-02-17 18:57:57	ID requested
⏵ 1	2020-02-17 19:01:48	announced

Lobingier BT, H, ü, ttenhain R, Eichel K, Miller KB, Ting AY, von Zastrow M, Krogan NJ, An Approach to Spatiotemporally Resolve Protein Interaction Networks in Living Cells. Cell, 169(2):350-360.e12(2017) [pubmed]

submitter keyword: GPCR, APEX, proximity biotinylation, protein-protein interactions

Ruth Huttenhain
contact affiliation	Department of Molecular and Cellular Pharmacology, UCSF
contact email	Ruth.huttenhain@ucsf.edu
lab head
Meena Choi
contact affiliation	Northeastern University
contact email	mnchoi67@gmail.com
dataset submitter

MassIVE dataset URI

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://massive.ucsd.edu/MSV000084967/