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Expanded metabolite coverage of Saccharomyces cerevisiae extract through improved chloroform/methanol extraction and tert-butyldimethylsilyl derivatization

Sakda Khoomrung (Institutionen för biologi och bioteknik, Systembiologi) ; José Luis Martinez Ruiz (Institutionen för biologi och bioteknik, Systembiologi) ; Stefan Tippmann (Institutionen för biologi och bioteknik, Systembiologi) ; Suwanee Jansa-Ard (Institutionen för biologi och bioteknik, Systembiologi) ; Marieke F. Buffing (Institutionen för biologi och bioteknik, Systembiologi) ; Raffaele Nicastro (Institutionen för biologi och bioteknik, Systembiologi) ; Jens B. Nielsen (Institutionen för biologi och bioteknik, Systembiologi)
Analytical Chemistry Research (2214-1812). Vol. 6 (2015), p. 9-16.
[Artikel, refereegranskad vetenskaplig]

We present an improved extraction and derivatization protocol for GC-MS analysis of amino/non-amino acids in Saccharomyces cerevisiae. Yeast cells were extracted with chloroform: aqueous-methanol (1:1, v/v) and the resulting non-polar and polar extracts combined and dried for derivatization. Polar and non-polar metabolites were derivatized using tert-butyldimethylsilyl (t-BDMS) dissolved in acetonitrile. Using microwave treatment of the samples, the derivatization process could be completed within 2 h (from >20 h of the conventional method), providing fully derivatized metabolites that contain multiple derivatizable organic functional groups. This results in a single derivative from one metabolite, leading to increased accuracy and precision for identification and quantification of the method. Analysis of combined fractions allowed the method to expand the coverage of detected metabolites from polar metabolites i.e. amino acids, organic acids and non-polar metabolites i.e. fatty alcohols and long-chain fatty acids which are normally non detectable. The recoveries of the extraction method was found at 88 ± 4%, RSD, N = 3 using anthranilic acid as an internal standard. The method promises to be a very useful tool in various aspects of biotechnological applications i.e. development of cell factories, metabolomics profiling, metabolite identification, 13C-labeled flux analysis or semi-quantitative analysis of metabolites in yeast samples.

Nyckelord: Derivatization, Extraction, Metabolomics, Saccharomyces cerevisiae

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Denna post skapades 2015-11-11. Senast ändrad 2017-01-17.
CPL Pubid: 225590


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Institutionen för biologi och bioteknik, Systembiologi


Bioinformatik och systembiologi

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