PXD000647

PXD000647 is an original dataset announced via ProteomeXchange.

Title	Functional analysis of novel Rab GTPases identified in the proteome of 2 purified Legionella-containing vacuoles from macrophages
Description	LCVs purified by immuno-magnetic separation and density gradient centrifugation (fraction 4) were resolved by 1D-SDS-PAGE, the gel lanes were excised in ten equidistant pieces and subjected to trypsin digestion. For the subsequent LC-MS/MS measurements, the digests were separated by reversed phase column chromatography using an EASYnLC apparatus with self-packed columns in a one-column setup. Following loading/ desalting in 0.1% acetic acid in water at a flow rate of 700 nL/min (maximum 220 bar), the peptides were separated by applying a binary non-linear gradient from 5-50% acetonitrile in 0.1% acetic acid over 70 min. The LC was coupled online to a LTQ-Velos Orbitrap mass spectrometer (Thermo Fisher, Bremen, Germany) at a spray voltage of 2.4 kV. After a survey scan in the Orbitrap (r = 30,000), MS/MS data were recorded for the twenty most intensive precursor ions in the linear ion trap. Singly charged ions were not taken into account for MS/MS analysis. The lock mass option was enabled throughout all analyses. After mass spectrometric measurement, MS data was converted into the mzxml format (version 3.1) by ReAdW version 4.3.1 and subsequently subjected to database searching via Sorcerer using Sequest (version 27, revision 11; SageN, Milptas, CA, USA) without charge state deconvolution and deisotoping performed. Database searching was performed either with a database of combined entries of L. pneumophila strain Philadelphia-1 (NC002942) and D. discoideum, or with a database of combined entries of L. pneumophila strain Philadelphia-1 and Mus musculus. Both databases were used as target/decoy databases with a list of common contaminants added (30739 and 64993 entries, respectively). Sequest was used assuming trypsinisation with a fragment ion mass tolerance of 1.00 Da and a search tolerance of 10 ppm for the overview scans. Oxidation of methionine was specified in Sequest as a variable modification. Scaffold (version 3.5.1, Proteome Software Inc., Portland, OR, USA) was used to filter and validate MS/MS-based peptide and protein identifications. Peptide identifications were accepted when spectra exceeded Xcorr values of 2.2, 3.3 and 3.8 for doubly, triply and quadruply peptides with deltaCN values of more than 0.1. Protein identifications are based on at least 2 unique peptides. Proteins that contained similar peptides and could not be differentiated based on MS/MS analysis alone were grouped to meet the principle of parsimony.
HostingRepository	PRIDE
AnnounceDate	2014-07-25
AnnouncementXML	Submission_2014-07-25_01:51:10.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Andreas Otto
SpeciesList	scientific name: Dictyostelium discoideum (Slime mold); NCBI TaxID: 44689; scientific name: Mus musculus (Mouse); NCBI TaxID: 10090; scientific name: Legionella pneumophila; NCBI TaxID: 446;
ModificationList	monohydroxylated residue
Instrument	LTQ Orbitrap Velos

Revision	Datetime	Status	ChangeLog Entry
0	2013-12-16 03:47:37	ID requested
1	2014-01-23 04:27:10	announced
⏵ 2	2014-07-25 01:51:11	announced	Updated project metadata.

Dataset with its publication pending

submitter keyword: Bacterial effector proteins, Dictyostelium discoideum, host-pathogen interactions, 18 Legionella, macrophage, pathogen vacuole, phosphoinositide lipids, GTPase, type IV secretion

Andreas Otto
contact affiliation	Institut fur Mikrobiologie
contact email	andreas.otto@uni-greifswald.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/2014/01/PXD000647

PRIDE project URI

• PRIDE
  1. PXD000647
     1. Label: PRIDE project
     2. Name: Functional analysis of novel Rab GTPases identified in the proteome of 2 purified Legionella-containing vacuoles from macrophages