氨基酸分析-同位素稀释质谱法准确定量胱抑素C重组蛋白

Absolute quantification of Cystatin C recombinant protein by amino acid analysis-isotope dilution mass spectrometry

目的:建立同位素稀释液相色谱串联质谱法进行氨基酸分析,准确定量人重组蛋白胱抑素C(Cys C)。方法:优化Cys C蛋白酸水解条件,将Cys C蛋白水解成氨基酸,以水解产物缬氨酸(Val)和苯丙氨酸(Phe)为研究对象,13C5-Val和13C9,15N-Phe为内标,根据括号法分别计算水解产物Val和Phe浓度,从而准确定量Cys C蛋白。结果:Cys C蛋白在130℃,6 mol/L HCl溶液中水解4 h可完全水解。以水解产物Val和Phe分别计算Cys C浓度为1.324 mg/g和1.328 mg/g,一致性较好,检测结果变异系数均小于7%(n=5),取二者平均值得到重组Cys C蛋白浓度为1.326 mg/g。结论:成功建立同位素稀释质谱法准确定量重组蛋白Cys C的氨基酸分析方法,有望为临床实验室蛋白质检测的溯源提供思路。

Objective：To establish an isotope dilution liquid chromatography tandem mass spectrometry method for the absolute quantification of human Cystatin C（Cys C） recombinant protein by amino acid analysis. Methods：All peptide bonds of Cys C were thoroughly broken into amino acids under optimized acidic hydrolysis conditions. The accurate quantification of Cys C was accomplished by setting the hydrolysate valine（Val） and phenylalanine（Phe） as analytes,13C5-Val and13C9and15N-Phe as internal standards, and applying the bracketing method to calculate the concentrations of Val and Phe.Results： The hydrolysis conditions optimized were 4 h with 6 mol/L HCl at 130℃. Cys C concentrations calculated from hydrolysate Val and Phe were 1.324 mg/g and 1.328 mg/g respectively, the average of which was 1.326 mg/g, showing good consistency and good repeatability with CV less than 7%（n=5）. Conclusion：The isotope dilution mass spectrometry method for the absolute quantification of human Cystatin C（Cys C） recombinant protein by amino acid analysis has been successfully established, which may provide ideas for solving the problem of traceability in protein quantification in clinical laboratory.