PXD000556

PXD000556 is an original dataset announced via ProteomeXchange.

Title	E. coli BW25113 and knock-out mutants (delta-pka, delta-arcA and delta-pka_arcA) LC MS/MS
Description	1ml of WT, delta-pka, delta-arcA and delta-pka delta-arcA culture broth was harvested at u=0.4 h-1, centrifuged (14000 x g for 1 min at 4 degrees C), pellet washed once with PBS, flash frozen with liquid N2 and kept at -80 degrees C until further processing. Frozen pellets were melted on ice after which 100 ug of sample biomass was mixed with 100 ug of SILAC-labeled E. coli biomass (internal standard) and frozen at -80 degrees C. Cell pellets were suspended in 100 uL SDS lysis buffer (4% SDS/100 mM TRIS-HCl pH 8/100 mM DTT) and heated at 95 degrees C for 15 min. Cell lysates were sonicated with ultrasound for a few pulses and pelleted by centrifugation. Cell lysates were digested with trypsin according to Filter Aided Sample Preparation protocol (FASP) (Wiśniewski et al. 2009) and purified with C-18 StageTips (Rappsilber et al. 2007). LC-MS/MS analysis was performed using an Agilent 1200 series nanoflow system (Agilent Technologies) connected to a LTQ Orbitrap mass-spectrometer (Thermo Electron, San Jose, CA, USA) equipped with a nanoelectrospray ion source (Proxeon, Odense, Denmark). Peptides were separated with a 240-minute gradient from 2 to 40% B (A: 0.5% acetic acid, B: 0.5% acetic acid/80% acetonitrile) using a flow-rate of 200 nl/min. Data analysis of raw MS-files was performed by the MaxQuant software package version 1.3.0.5 (Cox and Mann 2008). Peak lists were searched using the Andromeda search engine (built into MaxQuant) against an E. coli database (downloaded 09.04.2013 from http://www.uniprot.org/) which was supplemented with common contaminants (e.g human keratin, trypsin). Full tryptic specificity, a maximum of two missed cleavages and a mass tolerance of 0.5 Da for fragment ions was specified in the MaxQuant search. Carbamidomethylation of cysteine was set as a fixed modification, and methionine oxidation and protein N-terminal acetylation were set as variable modifications. The required false discovery rate was set to 1% for both peptide and protein levels and the minimum required peptide length was set to six amino acids. “Match between runs” option with a time window of 1.5 min was allowed.
HostingRepository	PRIDE
AnnounceDate	2014-07-25
AnnouncementXML	Submission_2014-07-25_01:44:46.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Karl Peebo
SpeciesList	scientific name: Escherichia coli; NCBI TaxID: 562; scientific name: Acyrthosiphon pisum (Pea aphid); NCBI TaxID: 7029;
ModificationList	methionine oxidation with neutral loss of 64 Da; S-carboxamidomethyl-L-cysteine; N-acetylated residue
Instrument	LTQ Orbitrap

Revision	Datetime	Status	ChangeLog Entry
0	2013-10-30 03:53:47	ID requested
1	2014-03-19 06:31:05	announced
⏵ 2	2014-07-25 01:44:47	announced	Updated project metadata.

Peebo K, Valgepea K, Nahku R, Riis G, Oun M, Adamberg K, Vilu R, Coordinated activation of PTA-ACS and TCA cycles strongly reduces overflow metabolism of acetate in Escherichia coli. Appl Microbiol Biotechnol, 98(11):5131-43(2014) [pubmed]

submitter keyword: E. coli, delta-pka, delta-arcA, delta-pka_arcA

Karl Peebo
contact affiliation	Competence Center of Food and Fermentation Technologies
contact email	karl@tftak.eu
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/2014/03/PXD000556

PRIDE project URI

• PRIDE
  1. PXD000556
     1. Label: PRIDE project
     2. Name: E. coli BW25113 and knock-out mutants (delta-pka, delta-arcA and delta-pka_arcA) LC MS/MS