液质联用多反应监测法定量目标多肽或蛋白质  

Quantification of Target Peptides or Proteins by Liquid Chromatography-Mass Spectrometry with Multiple Reaction Monitoring

作　　者：刘永福[1,2] 贾小芳[1] 腾珍林[1] 尹林[1] 刘保池[1] 张丽军[1,3] 

机构地区：[1]上海市公共卫生临床中心,上海201508 [2]郑州大学第一附属医院,郑州450052 [3]中南大学临床药理研究所,长沙410078

出　　处：《中国生物化学与分子生物学报》2012年第1期86-92,共7页Chinese Journal of Biochemistry and Molecular Biology

基　　金：中国博士后基金资助项目(No.20100471000;No.201104484);中国肝炎防治基金会王宝恩肝纤维化研究基金(No.20100031)~~

摘　　要：为建立优化的血浆内源性多肽提取方法,并且构建目标多肽和蛋白质的质谱定量方法,本研究考察了超滤法、有机溶剂沉淀法和固相萃取法对血浆内源性多肽的提取效果,并通过Tricine-SDS-PAGE对提取效果进行比较.通过液相色谱串联质谱多反应监测(MRM)分析,建立了多肽标准品ESAT-6定量方法,并将ESAT-6定量建立的液相色谱和质谱条件应用于蛋白质的定量,对多肽和蛋白质MRM定量的标准曲线进行了考察.Tricine-SDS-PAGE结果表明,乙腈沉淀法是最佳的血浆内源性多肽提取方法,低分子量的多肽可以得到很好的富集,且能有效地去除高分子蛋白质的污染.液相色谱串联质谱MRM法检测血浆内提取的多肽,标准曲线的线性较好,相关系数为0.999.另外,采用MRM法对胶内分离的蛋白质进行定量,标准曲线的线性相关系数为0.995.综上所述,本研究构建了一种简单有效的血浆多肽提取方法,通过液质联用MRM法成功地实现了目标多肽和蛋白质定量测定.该定量方法可以推广应用于复杂样品中的多肽和蛋白质的定量分析.This study was to establish a modified plasma peptide extraction method and to develop a mass spectrometry quantification method for target peptides or proteins.Three techniques of ultrafiltration,organic solvent extraction and solid phase extraction were used to extract peptides from plasma samples.Tricine-SDS-PAGE was used to determine the extraction efficiency.Peptide and protein quantification were completed by liquid chromatography combined with mass spectrometry in multiple reaction monitoring（MRM） mode.The Tricine-SDS-PAGE results showed that acetonitrile（ACN） precipitation was the most efficient approach for low molecular plasma peptides enrichment besides for excluding the high molecular protein contaminations.The correlation coefficients of the standard curves were 0.995 and 0.999 in the quantification of peptide or protein standards by liquid chromatography-mass spectrometry（LC-MS）,respectively.In conclusion,we successfully established a simple and efficient plasma peptide extraction method and a subsequent LC-MS MRM method for peptide and protein quantifications.These methods might be applied to the quantification of target peptides or proteins in complex samples.

关 键 词：多肽 蛋白质 多反应监测 定量 液相色谱串联质谱 

分 类 号：Q5-33[生物学—生物化学]