PXD000415

PXD000415 is an original dataset announced via ProteomeXchange.

Title	Human Hippo pathway , LC-MSMS
Description	The Hippo pathway, which is conserved from Drosophila to mammals, has been recognized as a tumor suppressor signaling pathway governing cell proliferation and apoptosis, two key events involved in organ size control and tumorigenesis. Although several upstream regulators, the conserved kinase cascade and key downstream effectors including nuclear transcriptional factors have been defined, the global organization of this signaling pathway is not been fully understood. Thus, we conducted a proteomic analysis of human Hippo pathway, which revealed the involvement of an extensive protein-protein interaction network in this pathway. Our data suggest that 550 interactions within 343 unique protein components constitute the central protein-protein interaction landscape of human Hippo pathway. Our study provides a glimpse into the global organization of Hippo pathway, reveals previously unknown interactions within this pathway, and uncovers new potential components involved in the regulation of this pathway. Understanding these interactions will help us further dissect the Hippo signaling-pathway and extend our knowledge of organ size control. Mass spectrometry data anaylsis: Excised gel bands were cut into approximately 1 mm3 pieces. Gel pieces were then subjected to in-gel trypsin digestion and dried. Samples were reconstituted in 5 ul of HPLC solvent A (2.5% acetonitrile, 0.1% formic acid). A nano-scale reverse-phase HPLC capillary column was created by packing 5 um C18 spherical silica beads into a fused silica capillary (100 um inner diameter x 20 cm length) with a flame-drawn tip. After equilibrating the column each sample was loaded via a Famos autosampler (LC Packings, San Francisco CA) onto the column. A gradient was formed and peptides were eluted with increasing concentrations of solvent B (97.5% acetonitrile, 0.1% formic acid). As peptides eluted they were subjected to electrospray ionization and then entered into an LTQ Velos ion-trap mass spectrometer (ThermoFisher, San Jose, CA). Peptides were detected, isolated, and fragmented to produce a tandem mass spectrum of specific fragment ions for each peptide. Peptide sequences (and hence protein identity) were determined by matching protein databases with the acquired fragmentation pattern by the software program, SEQUEST (ver. 28). (ThermoFisher, San Jose, CA). Enzyme specificity was set to partially tryptic with 2 missed cleavages. Modifications included carboxyamidomethyl (cysteines, fixed) and oxidation (methionine, variable). Mass tolerance was set to 2.0 for precursor ions and 1.0 for fragment ions. The database searched was the Human IPI databases version 3.6. Because we used HEK293 cells the Human IPI database was used. The number of entries in the database was 160,900 which included both the target (forward) and the decoy (reversed) human sequences. Spectral matches were filtered to contain less than 1% FDR at the peptide level based on the target-decoy method. Finally, only tryptic matches were reported and spectral matches were manually examined. When peptides matched to multiple proteins, the peptide was assigned so that only the most logical protein was included (Occam's razor). This same principle was used for isoforms when present in the database.
HostingRepository	PRIDE
AnnounceDate	2014-07-25
AnnouncementXML	Submission_2014-07-25_01:32:56.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Xu Li
SpeciesList	scientific name: Homo sapiens (Human); NCBI TaxID: 9606;
ModificationList	monohydroxylated residue; iodoacetic acid derivatized residue
Instrument	LTQ Orbitrap

Revision	Datetime	Status	ChangeLog Entry
0	2013-08-15 02:12:19	ID requested
1	2013-10-17 05:58:56	announced
1	2013-10-17 06:19:17	announced
⏵ 2	2014-07-25 01:32:56	announced	Updated project metadata.

Dataset with its publication pending

submitter keyword: Tandem affinity purification, protein interactome, Hippo pathway

Xu Li
contact affiliation	Experimental Radiation Oncology
contact email	xulimda@gmail.com
dataset submitter

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PRIDE project URI

• PRIDE
  1. PXD000415
     1. Label: PRIDE project
     2. Name: Human Hippo pathway , LC-MSMS