基于液相色谱-同位素稀释质谱法测定氨基酸的蛋白质定量方法

Protein Quantitative Method Based on Determination of Amino Acid by LC-ID-MS

为定量分析蛋白质,建立蛋白质水解液中氨基酸的液相色谱-同位素稀释质谱分析方法。蛋白质在6 mol/L盐酸溶液存在下130℃水解,经Hypercarb多孔石墨碳色谱柱、0.1%全氟戊酸水溶液/乙腈流动相体系分离,选择脯氨酸、缬氨酸、亮氨酸、异亮氨酸、苯丙氨酸5种较为稳定的氨基酸,以其相应的稳定同位素标记物为内标括号法定量,结合蛋白质的氨基酸序列信息实现对蛋白质的绝对定量。结果表明,5种氨基酸在10~1000 ng/mL范围内线性关系良好(R^(2)>0.995),检出限和定量限分别在0.02~0.07 ng/mL和0.06~0.2 ng/mL之间,3个不同质量水平下经水解处理的回收率在97.0%~102.4%之间、相对标准偏差在1.1%~3.0%之间。对新型冠状病毒(新冠病毒)N蛋白人源IgG单克隆抗体有证标准物质的分析结果和新冠病毒N蛋白国家计量比对的测量结果满意,证明该方法具有足够的准确度。

In order to quantitate protein,a liquid chromatography-isotope dilution mass spectrometry method for the analysis of amino acids in protein hydrolysate was established.The protein was hydrolyzed at 130℃in the presence of 6 mol/L hydrochloric acid solution,and separated by Hypercarb porous graphite carbon chromatography column and 0.1%perfluoropentanoic acid aqueous solution/acetonitrile mobile phase system.Five stable amino acids(proline,valine,leucine,isoleucine and phenylalanine)were selected for quantitative determination with their corresponding stable isotope markers as internal standard,and the absolute quantitative determination of protein was achieved by combining protein sequence information.The linear relationship of the five amino acids is good within the range of 10-1000 ng/mL(R^(2)>0.995),the limit of detection and the limit of quantification are between 0.02-0.07 ng/mL and 0.06-0.2 ng/mL,respectively.The recovery at three different levels is between 97.0%-102.4%,and the relative standard deviation is between 1.1%-3.0%.The analysis results of human IgG monoclonal antibody to nucleocapsid protein solution reference material of 2019 Novel Coronavirus and the measurement results of national metrological comparison of 2019-nCovid nucleocapsid protein are satisfactory,which proves that the method has sufficient accuracy for protein determination.