Dataset Information

E. coli BW25113 and knock-out mutants (delta-pka, delta-arcA and delta-pka_arcA) LC MS/MS

ABSTRACT: 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.

INSTRUMENT(S): LTQ Orbitrap

ORGANISM(S): Acyrthosiphon Pisum (pea Aphid) Escherichia Coli

SUBMITTER: Karl Peebo

LAB HEAD: Karl Peebo

PROVIDER: PXD000556 | Pride | 2014-03-19

REPOSITORIES: Pride

ACCESS DATA

Dataset's files

Source:

			Action	DRS
	130329_07_Orbi1_SK_SER_F200_S1_P5.raw	Raw
	130402_05_Orbi1_SK_SER_pka1.raw	Raw
	130402_06_Orbi1_SK_SER_pka2.raw	Raw
	130402_07_Orbi1_SK_SER_pka3.raw	Raw
	130403_17_Orbi1_SK_SER_pka_arcA1.raw	Raw

Items per page:

1 - 5 of 11

Publications

Coordinated activation of PTA-ACS and TCA cycles strongly reduces overflow metabolism of acetate in Escherichia coli.

Peebo Karl K Valgepea Kaspar K Nahku Ranno R Riis Gethe G Oun Mikk M Adamberg Kaarel K Vilu Raivo R

Applied microbiology and biotechnology 20140315 11

Elimination of acetate overflow in aerobic cultivation of Escherichia coli would improve many bioprocesses as acetate accumulation in the growth environment leads to numerous negative effects, e.g. loss of carbon, inhibition of growth, target product synthesis, etc. Despite many years of studies, the mechanism and regulation of acetate overflow are still not completely understood. Therefore, we studied the growth of E. coli K-12 BW25113 and several of its mutant strains affecting acetate-related ...[more]

PMID: 24633370

Similar Datasets

Project description:Two datasets: StREM1.3: methyl-beta cyclodextrin-dependent distribution of StREM1.3 to detergent resistant and detergent soluble membrane fractions. SLAH3: localization of SLAH3 to detergent resistant and detergent soluble fractions dependent on co-expression with CPK21 and ABI1. Data analysis: Protein identiﬁcation and ion intensity quantitation was carried out by MaxQuant version 1.3.0.5. Spectra were matched against the Arabidopsis proteome (TAIR10, 35,386 entries) using Andromeda. Thereby, carbamidomethylation of cysteine was set as a ﬁxed modiﬁcation; oxidation of methionine as well as phosphorylation of serine, threonine, and tyrosine was set as variable modiﬁcations. Because label-free quantitation based on ion intensities was used, multiplicity was set to 1. Mass tolerance for the database search was set to 20 ppm on full scans and 0.5 Da for fragment ions. Retention time matching between runs was chosen within a time window of 2 min. Peptide false discovery rate (FDR), protein FDR was set to 0.01, and site FDR was set to 0.05. Contaminants and reverse hits identiﬁed by MaxQuant were excluded from further analys Protein identiﬁcation and ion intensity quantitation was carried out by MaxQuant version 1.3.0.5. Spectra were matched against the Arabidopsis proteome (TAIR10, 35,386 entries) using Andromeda. Thereby, carbamidomethylation of cysteine was set as a ﬁxed modiﬁcation; oxidation of methionine as well as phosphorylation of serine, threonine, and tyrosine was set as variable modiﬁcations. Because label-free quantitation based on ion intensities was used, multiplicity was set to 1. Mass tolerance for the database search was set to 20 ppm on full scans and 0.5 Da for fragment ions. Retention time matching between runs was chosen within a time window of 2 min. Peptide false discovery rate (FDR), protein FDR was set to 0.01, and site FDR was set to 0.05. Contaminants and reverse hits identiﬁed by MaxQuant were excluded from further analysis. PXD000211 is also assoicated with the same study/paper.

Project description:MicroPoly(A)Pure kits (Ambion) were used to prepare mRNA from P. salmonis-infected and control Atlantic salmon head kidney. Head kidney mRNA samples were prepared from pooled (n = 10) infected or control kidney. Approximately 200 ng of infected or control head kidney mRNA was used as template in aRNA synthesis reactions. Two infected head kidney samples were pooled to yield 8.9 ug of aRNA, and a single control head kidney sample contained 8.4 ug of aRNA. Infected and control head kidney aRNA samples were visualized on agarose gels to check quality and quantity (data not shown). Except for the amounts of aRNA and reverse transcription (RT) primer used, head kidney target labeling reactions and microarray hybridizations were identical. Head kidney target syntheses used 2 ug of aRNA and 2 ug of random primers (Roche). RT primers were mixed with aRNA samples, and nuclease-free H2O (Invitrogen) was added to make 10 ul total volume. Samples were incubated at 70 degrees C for 4 min, then placed on ice. RT reactions included 50 mM Tris HCl pH 8.3, 75 mM KCl, 3 mM MgCl2, 10 mM DTT, 0.5 mM each dATP, dCTP, and dGTP, 0.2 mM dTTP, 0.05 mM Cy3-dUTP or Cy5-dUTP (Amersham), 40U RNAguard ribonuclease inhibitor (Amersham), and 400U Superscript II Rnase H- reverse transcriptase (Invitrogen). RT reactions were incubated at 42 degrees C for 2.5 h, followed by addition of 0.025U RNase A (Sigma) and 1.5U RNase H (New England BioLabs) and incubation at 37 degrees C for 30 min. Labeled targets were cleaned using the Qiaquick PCR purification kit (Qiagen) and precipitated, with 300 mM sodium acetate, 1 ul of 20 ug/ul glycogen (Invitrogen), and 2.5 volumes of 100% ethanol, at -20 degrees C overnight. Each labeled target was recovered by centrifugation (14,000 rpm, 4 degrees C, 1 h), washed in 70% ethanol, air-dried, and resuspended in 60 ul of hybridization buffer: 50% deionized formamide, 5X SSC, 0.1% SDS, 5 ul of 5 ug/ul oligo dT (5’T18[Q-N]3’), 5 ul of 2 ug/ul BSA (Pierce), and 2 ul of 10 ug/ul sonicated human placental DNA (Sigma). Targets were incubated at 96 degrees C for 3 min, and then at 65 degrees C until applied to microarrays. Microarrays were prepared for hybridization by washing 2 X 5 min in 0.1% SDS, washing 5 X 1 min in MilliQ H2O, immersing 3 min in 95 degrees C MilliQ H2O, and drying by centrifugation (2000 rpm, 5 min, in 50 ml conical tube). Microarray hybridizations were run in the dark under HybriSlips hybridization covers (Grace Biolabs) in slide hybridization chambers (Corning) submerged in a 45 degrees C water bath for 16 h. Coverslips were floated off in 45 degrees C (1X SSC, 0.2% SDS), and arrays were washed 1 X 10 min in 45 degrees C (1X SSC, 0.2% SDS), 3 X 4 min in room temperature (0.1X SSC, 0.1% SDS), and 4 X 4 min in room temperature 0.1X SSC. Slides were dried by centrifugation as before. Fluorescent images of hybridized arrays were acquired immediately at 10 um resolution using ScanArray Express (PerkinElmer). The Cy3 and Cy5 cyanine fluors were excited at 543 nm and 633 nm respectively, and the same laser power (90%) and photomultiplier tube (PMT) settings were used for all slides in a study (PMT 75 Cy3, PMT 63 or 64 Cy5 for head kidney study). Fluorescent intensity data were extracted from TIF images using Imagene 5.5 software (Biodiscovery). Keywords: other