高效液相色谱-柱后同位素稀释质谱法定量分析人血清中转铁蛋白及白蛋白  

Absolute Quantification of Transferrin and Albumin in Human Serum by Species-unspecific Isotope Dilution-Liquid Chromatography-Inductively Coupled Plasma Mass Spectrometry

作　　者：张丹[1,2] 冯流星[2] 王军[2] 熊金平[1] 

机构地区：[1]北京化工大学,材料科学与工程学院,北京100029 [2]中国计量科学研究院,北京100013

出　　处：《分析化学》2014年第2期179-185,共7页Chinese Journal of Analytical Chemistry

基　　金：国家质检总局公益性行业科研专项(No.201110008)资助项目

摘　　要：建立了高效液相色谱(HPLC)与电感耦合等离子体质谱(ICP-MS)联用,同时结合柱后同位素稀释法,通过直接测定硫及铁元素,实现蛋白质绝对定量的分析方法。选取分子量不同的4种标准蛋白:转铁蛋白、β乳球蛋白、肌红蛋白和溶菌酶作为混合蛋白,34S或54Fe同位素稀释剂与液相色谱洗脱液经三通混合后,在线进入ICP-MS检测。根据同位素稀释法公式及蛋白中硫、铁的含量计算混合蛋白中每种蛋白的浓度,与天平称量结果一致,对方法进行了验证。将方法应用到人血清转铁蛋白、白蛋白的定量分析,针对两种蛋白含量差别大导致的信号差异,通过改变同位素稀释剂的流速,成功测定了人血清中转铁蛋白、白蛋白的含量。采用34S及54Fe同位素稀释剂分别定量的血清中转铁蛋白浓度为(2.35±0.01)g/L和(2.22±0.13)g/L,结果基本吻合。方法精密度RSD均小于10%,可用于基体样品中含硫蛋白及金属蛋白的定量分析。A method based on species-unspecific isotope dilution-high performance liquid chromatography coupled with inductively coupled plasma mass spectrometry （HPLC-ID-ICP-MS） was established for the absolute quantification of proteins by measuring sulfur and iron. Four standard proteins including transferrin,lactoglobulin, myoglobin and lysozyme were separated and on-line determined via sulfur or iron by enriched 34S or SaFe isotopic solutions continuously mixing with the eluate from liquid chromatography. Absolute quantification of proteins was calculated by the isotope dilution equation and sulfur or iron content in proteins. The methodology was validated by comparing with gravimetric analysis results. Furthermore, this method was successfully applied to the quantification of transferrin and albumin in human serum by means of changing the flow rate of isotopic solution. The measured results of transferrin via sulfur and iron were （2.35±0.01） g/L, （2.22±0.13） g/L, respectively, which was in good agreement and the RSDs were all within 10%. The method can be used to quantify sulfur containing proteins and metalloproteins in biological samples.

关 键 词：同位素稀释 蛋白质定量 电感耦合等离子体质谱 转铁蛋白 白蛋白 

分 类 号：R341[医药卫生—基础医学] O657.63[理学—分析化学]