液相色谱-同位素稀释串联质谱法定量分析重组N末端B型利钠肽原  

Quantitative Analysis of Recombinant N-terminal Pro-brain Natriuretic Peptide by Liquid Chromatography Coupled with Isotope Dilution Mass Spectrometry

作　　者：孙泽朋 王洪彬[1] 陈金超 宋德伟[2,3] 李红梅 肖鹏[2,3] SUN Ze-Peng;WANG Hong-Bin;CHEN Jin-Chao;SONG De-Wei;LI Hong-Mei;XIAO Peng(College of Biotechnology,Tianjin University of Science and Technology,Tianjin 300457,China;National Institute of Metrology,Beijing 100029,China;Key Laboratory of Chemical Metrology and Applications on Nutrition and Health for State Market Regulation,Beijing 100029,China)

机构地区：[1]天津科技大学生物工程学院,天津300457 [2]中国计量科学研究院,北京100029 [3]国家市场监管重点实验室(营养与健康化学计量及应用),北京100029

出　　处：《分析化学》2023年第1期120-129,共10页Chinese Journal of Analytical Chemistry

基　　金：国家重点研发计划项目(No.2019YFF0216505);中国计量科学研究院基本科研业务费项目(Nos.AKYZD2115-2,AKYGH2002)资助。

摘　　要：N末端B型利钠肽原(NT-proBNP)是心力衰竭临床诊断的重要生物标志物之一。目前,国际上尚没有NT-proBNP标准物质,无法在溯源链顶端开展量值传递,不利于下游测量结果的统一和对比。为了实现NT-proBNP的标准化检测,本研究针对重组NT-proBNP开展了溯源技术研究。首先采用蛋白免疫印迹和质谱法对被测物进行了分析。在绝对定量分析方法研究中,采用两种同位素稀释质谱法对NT-proBNP样品进行了定值。结果表明,重组NT-proBNP具有良好的抗体结合活性,并且分子量与理论值一致,可用于后续的定量分析方法研究。在氨基酸同位素稀释质谱法中,NT-proBNP水解48 h后达到平衡,通过对水解样品中缬氨酸、异亮氨酸和精氨酸的测量,计算得到NT-proBNP溶液的质量分数为79.62μg/g(RSD=0.89%);在肽段同位素稀释质谱法中,当NT-proBNP与蛋白酶质量比为25∶1、酶解24 h后,通过检测两条特征肽段,计算得到NT-proBNP溶液质量分数为76.04μg/g(RSD=1.85%)。两种方法测量结果基本一致。由于定量氨基酸是具有计量学溯源性的国家一级标准物质,因此基于本方法的NT-proBNP测量结果具备了溯源性,为标准物质的研制奠定了基础。N-terminal pro-brain natriuretic peptide(NT-proBNP)is an essential biomarker for diagnosis of heart failure.Currently,since reference materials of NT-proBNP are unavailable globally,value transfer activities are hard to implement from the higher level to the end-users,which makes terminal clinical testing results inconsistent and incomparable.To achieve standard detection of NT-proBNP,in this work,recombinant NT-proBNP was chosen as a target during traceable approach development.Firstly,the immune capacity and relative molecular weight were tested and varied through Western Blot assay and mass spectrometric methods.Next,two kinds of isotope dilution mass spectrometry were adopted for the absolute quantification of NT-proBNP.The results suggested that the protein hydrolysis reaction reached equilibrium in 48 h.NT-proBNP was calculated to be 79.62μg/g with an relative standard deviation(RSD)of 0.89%by detection of valine,isoleucine and arginine.On the other hand,the isotope dilution mass spectrometry method was used to analyze signature peptides produced by enzymatic digestion.NT-proBNP was calculated to be 76.04μg/g with an RSD of 1.85%by detection of the two peptides under the conditions of enzymatic digestion for 24 h and at mass ratio of 25∶1 of NT-proBNP to protease.The quantitative analysis results from the two methods were consistent with each other.As the amino acids applied in quantifications were SI-traceable primary reference materials,all the results were also traceable.Such approaches were candidates for NT-proBNP reference material development.

关 键 词：心力衰竭标志物 N末端B型利钠肽原 定量分析 同位素稀释质谱法 溯源性 

分 类 号：O657.63[理学—分析化学] R446.1[理学—化学]