Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA274761

Local Sample ID:	Liquid Media 2-02-4787
Subject ID:	SU002836
Subject Type:	Yeast
Subject Species:	Candida albicans
Taxonomy ID:	5476

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Sample Preparation:

Sampleprep ID:	SP002842
Sampleprep Summary:	The extraction method started by adding add 400 µl of protease inhibitor (dissolved in lysis buffer) to each pellet. Then were mixed and incubated for 10 minutes in room temperature, vortex each sample for 30 seconds. Cell lysates then were sonicated using COPLEY sonicator (Qsonica, Newtown, CT, USA) at 30% AMP for 30 seconds for 3 rounds until pellets dissolved homogenously. The samples were transferred to a new Eppendorf tube then centrifuge at 14000 rpm for 5 minutes. The supernatants, which contain the proteins and metabolites, were transferred to a new Eppendorf. Afterwards, 400µl of methanol were added to the transferred supernatant, followed by 300 µl of chloroform, then vortexed. Centrifuged again at 14000 rpm for 5 minutes to get eventually an upper layer (contains metabolites), an interphase of white disk (proteins) and lower layer. The supernatants that contained the metabolites were transferred carefully to a new tube, without disturbing the white disk. The interphase and the lower phase were mixed with 300µl of methanol. Vortexed well then centrifuged for 5 minutes at 14000 RPM and the new supernatants were added to the previously collected one. Later, the metabolites were completely dried using EZ-2 Plus (GeneVac, Ipswich, UK) at 45 ºC. After that, the samples were reconstituted in 200 µl of 0.1% Formic acid with water v/v. Finally, samples were filtered using 0.22µm filters and transferred into micro-insert to be analysed by TIMS-TOF MS.

Sampleprep ID:

SP002842

Sampleprep Summary:

The extraction method started by adding add 400 µl of protease inhibitor (dissolved in lysis buffer) to each pellet. Then were mixed and incubated for 10 minutes in room temperature, vortex each sample for 30 seconds. Cell lysates then were sonicated using COPLEY sonicator (Qsonica, Newtown, CT, USA) at 30% AMP for 30 seconds for 3 rounds until pellets dissolved homogenously. The samples were transferred to a new Eppendorf tube then centrifuge at 14000 rpm for 5 minutes. The supernatants, which contain the proteins and metabolites, were transferred to a new Eppendorf. Afterwards, 400µl of methanol were added to the transferred supernatant, followed by 300 µl of chloroform, then vortexed. Centrifuged again at 14000 rpm for 5 minutes to get eventually an upper layer (contains metabolites), an interphase of white disk (proteins) and lower layer. The supernatants that contained the metabolites were transferred carefully to a new tube, without disturbing the white disk. The interphase and the lower phase were mixed with 300µl of methanol. Vortexed well then centrifuged for 5 minutes at 14000 RPM and the new supernatants were added to the previously collected one. Later, the metabolites were completely dried using EZ-2 Plus (GeneVac, Ipswich, UK) at 45 ºC. After that, the samples were reconstituted in 200 µl of 0.1% Formic acid with water v/v. Finally, samples were filtered using 0.22µm filters and transferred into micro-insert to be analysed by TIMS-TOF MS.