气相色谱-质谱分析血清代谢组前处理中的除蛋白及衍生方法

摘要

The effects of different deproteinization and derivatization methods in serum sample preparation on metabolome were compared by gas chromatography-mass spectrometry.The relative standard derivations (RSD) of peak area and information content of profile were calculated.The principal components analysis (PCA),qualitative and quantitative analysis of serum samples and quality control samples (QC) were done.The RSD of QC and serum samples was less than 10％.The result of information content and PCA showed that these serum preparation methods were stable and reliable,The PCA result of profile produced by four preparation methods showed that the differences existed in serum metabolome when the samples were deproteinized and derivatizated by different reagents in preparation procedures.The influence of derivatization reagents on serum metabolome was great than that of deproteinization reagents.The information content of samples derivatizated by N-methyl-N-(trimethylsilyl) trifluoroacetamide (MSTFA) (8.9) was higher than that of samples derivatizated by BSTFA (8.7).Forty-four metabolites were obtained by qualitative analysis.Five metabolites were unique to samples derivatizated by MSTFA,and six metabolites were unique to samples derivatizated by N,O-Bis (trimethylsilyl) trifluoroacetamide (BSTFA).We proposed that when polar amino acids and short-chain fatty acids were concerned,BSTFA was recommended as derivatization reagent,and when nonpolar amino acids and long-chain fatty acids were concerned,MSTFA was recommended as derivatization reagent.%采用气相色谱-质谱联用技术(GC-MS),对代谢组学研究中血清样本的前处理方法进行考察,研究蛋白去除和衍生化两个步骤对血清代谢物分析的影响.对血清样本和质控(Quality control,QC)样本的图谱进行峰相对标准偏差(RSD)计算,全谱主成分分析(Principal components analysis,PCA)和信息量的计算以及代谢物的定性定量分析.结果表明,QC样本和血清平行样的峰面积RSD均小于10％,PCA分析结果及信息量计算结果都表明这些前处理方法重复性及可靠性良好,适用于代谢组学研究.4种前处理方法所得代谢图谱的PCA分析结果表明不同除蛋白试剂和衍生化试剂对代谢物谱存在不同的影响,其中衍生化试剂的影响大于除蛋白试剂的影响.信息量计算显示MSTFA组图谱的信息量(约8.9)大于BSTFA组的信息量(约8.7).对血清样本定性共得到44种代谢物,MSTFA组血清样本特有代谢物5种,BSTFA血清样本特有代谢物6种.对定性结果进行分析后建议:当需要研究极性氨基酸和短链脂肪酸时,采用BSTFA作衍生化试剂较合适；而当需要研究非极性氨基酸和长链脂肪酸时,采用MSTFA作衍生化试剂较合适.