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MB Sample ID: SA100964

Local Sample ID:CSH4-1
Subject ID:SU001456
Subject Type:Bacteria
Subject Species:Escherichia coli
Taxonomy ID:562

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Subject:

Subject ID:SU001456
Subject Type:Bacteria
Subject Species:Escherichia coli
Taxonomy ID:562

Factors:

Local Sample IDMB Sample IDFactor Level IDLevel ValueFactor Name
CSH4-1SA100964FL014206-Grown in presence of NaCl (mM)

Collection:

Collection ID:CO001451
Collection Summary:3ml of LB was inoculated with 0.1% inoculum from overnight grown culture and grown till OD600~0.8 at 37°C, 200 rpm. Cells equivalent to 0.1 OD were harvested at 14000 rpm, 1 min, 4°C.
Sample Type:Bacterial cells

Treatment:

Treatment ID:TR001471
Treatment Summary:CSH4 and WG350 strains were transformed with the mutant GFP clones and subjected to osmotic stress with 350mM NaCl. The pool of mutants buffered under osmotic stress in WG350 was sorted and single clones of GFP were picked from here and checked for their fluorescence in presence and absence of osmotic stress.

Sample Preparation:

Sampleprep ID:SP001464
Sampleprep Summary:Cell pellets were extracted with 200ul of chilled extraction solvent (80% MetOH in MS grade water containing 1ng/µl PIPES and U13C-U15N-glutamine as internal standard) was added to the pellet, mixed and incubated in ice for 5 minutes for quenching metabolism. This is followed by sonication in water bath for 15 min at 4°C with intermittent vortexing. Metabolites were collected in the supernatant by centrifugation at 14000 rpm for 5 min at 4°C. Previous step was repeated twice by adding 100µl 80% chilled solvent each time to increase metabolite yield and polled extracts were stored in -80°C refrigerator till further analysis.

Combined analysis:

Analysis ID AN002303
Analysis type MS
Chromatography type Reversed phase
Chromatography system Thermo Accela 1250
Column Thermo Accucore C18 (100 2.1mm, 2.6um)
MS Type ESI
MS instrument type Orbitrap
MS instrument name Thermo Q Exactive Orbitrap
Ion Mode NEGATIVE
Units Peak Intensity

Chromatography:

Chromatography ID:CH001692
Chromatography Summary:The acetonitrile:water extracts from parasites were dried under nitrogen flow and resuspended in 200 µls of water:methanol (97:3) containing 10 mM tributylamine and 15 mM acetic acid. A Thermo Accucore C18 column with a bed volume of 150 mm x 2.1 mm and 2.6 µ particle size was used. A solvent system composed of water buffered with 0.1 % formic acid (buffer A) and acetonitrile (buffer B), was used on a 20 minute gradient run with a flow rate of 200 µl/min as follows- hold at 10% acetonitrile for 30 seconds and gradually ramp up to 15%, 20%, 50%, 60% and 90% acetonitrile by 3, 6, 10, 12, 13 minutes, hold at 90% acetonitrile till 15 minutes, ramp down to 10% acetonitrile by 15.5 minutes and hold till 20 minutes.
Instrument Name:Thermo Accela 1250
Column Name:Thermo Accucore C18 (100 2.1mm, 2.6um)
Chromatography Type:Reversed phase

MS:

MS ID:MS002146
Analysis ID:AN002303
Instrument Name:Thermo Q Exactive Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:LC-MS analysis was done using a Exactive Orbitrap mass spectrometer, coupled to an Accela U-HPLC and HTC PAL autosampler. The mass spectrometer was run in negative mode, scanning a mass-charge ratio (m/z) range of 85-1000. The RAW file output from the mass spectrometer was converted from the profile mode into centroid mode using the ReAdW or Proteowizard program and further analyzed using the ElMAVEN program. Data from replicate samples for each time point was aligned within MAVEN and ion chromatograms were extracted for each compound to within a 10 PPM window of the expected m/z value. Peaks were detected from these ion chromatograms and their quality was ascertained using default settings available in MAVEN. Metabolites were identified by matching the retention times as well as the m/z values to >99% pure commercial standards for which in-house calibration was done. Grouped peaks from replicate samples for all time points were matched to the expected retention time of standards, and the peaks with a quality score of at least 0.5 were hand picked for metabolites of interest. Signals obtained from blank runs were used for noise correction and only peaks with a signal intensity of at least 1000 counts were considered.
Ion Mode:NEGATIVE
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