Expanded metabolite coverage of Saccharomyces cerevisiae extract through improved chloroform/methanol extraction and tert-butyldimethylsilyl derivatization
Sakda Khoomrung , Jose L. Martinez , Stefan Tippmann , Suwanee Jansa-Ard , Marieke F. Buffing , Raffaele Nicastro , Jens Nielsen
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引用次数: 17
Abstract
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.
我们提出了一种改进的GC-MS分析酿酒酵母中氨基酸/非氨基酸的提取和衍生化方案。用氯仿:水-甲醇(1:1,v/v)提取酵母细胞,将得到的非极性和极性提取物组合并干燥衍生化。极性代谢物和非极性代谢物用乙腈溶解的叔丁基二甲基硅基(t-BDMS)衍生化。使用微波处理样品,衍生化过程可在2小时内完成(常规方法为20小时),提供含有多个衍生化有机官能团的完全衍生化代谢物。这导致从一种代谢物中产生单一衍生物,从而提高了该方法鉴定和定量的准确性和精密度。对组合组分的分析使该方法扩大了从极性代谢物(即氨基酸、有机酸和非极性代谢物,即脂肪醇和长链脂肪酸)中检测到的代谢物的覆盖范围,这些代谢物通常是不可检测的。以邻氨基苯甲酸为内标,提取回收率为88±4%,RSD, N = 3。该方法有望在生物技术应用的各个方面成为非常有用的工具,例如细胞工厂的开发,代谢组学分析,代谢物鉴定,酵母样品中代谢物的13c标记通量分析或半定量分析。