摘要
建立了聚丙烯酰胺凝胶电泳（ PAGE）-激光烧蚀进样电感耦合等离子体质谱（ LA-ICP-MS）与非特异性同位素稀释法联用技术，通过测定蛋白条带上硫元素的含量，实现了人血清中蛋白的定量分析.血清电泳分离条件为：分离胶浓度（质量分数）为7.5%，浓缩胶浓度为4%，电泳电压100 V.为优化激光烧蚀实验条件，以LA-ICP-MS测得不同浓度硫富集凝胶中32 S的信号强度，在1~400μg/g范围内呈现良好的线性关系， R2=0.993.采用外标法对血清中的转铁蛋白和白蛋白进行了相对定量分析.此外，重点采用“电泳后同位素稀释”方法实现了蛋白条带上34 S稀释剂的加入，对血清中转铁蛋白和白蛋白进行了绝对定量分析；采用人血清蛋白标准物质ERM-DA470/IFCC对建立的方法体系进行了验证，结果表明2种蛋白的定量结果与标准值吻合，方法的准确性好，且测量精度优于外标法.
A methodology based on species-unspecific isotope dilution-gel electrophoresis-laser ablation cou-pled with inductively coupled plasma mass spectrometry（ GE-LA-ID-ICP-MS） was established for the absolute quantification of proteins in human serum by measuring sulfur. The electrophoresis separation condition of hu-man serum were 7. 5% separating gel, 4% stacking gel, 100 V until bromophenol blue dye had migrated to the bottom of gel. The average signal intensity of 32 S at different concentrations of sulfur enriched gels（1-400μg/g） showed a good linear relationship. The relative quantitative analysis of transferrin and albumin in hu-man serum was investigated using external standard method. In addition, the created species-unspecific iso-tope dilution in GE-LA-ICP-MS had added 34 S spike signals into gels successfully by mixing sulfur spike with protein strips. According to isotope dilution equation, the method was applied to a fast, accurate absolute quantification of transferrin and albumin in human serum. The methodology was validated by comparing with the quantitative analysis of a serum protein certified reference material（ ERM-DA470/IFCC） . The results con-firmed that determined concentration of transferrin and albumin fitted well with the certified value and the methodology had good accuracy and better measurement precision than external standard method.
出处
《高等学校化学学报》
SCIE
EI
CAS
CSCD
北大核心
2014年第9期1889-1895,共7页
Chemical Journal of Chinese Universities
基金
国家质检总局公益性行业科研专项(批准号:201110008)
中国计量科学研究院基本科研业务费(批准号:AKY1403-14)
科技部基础性专项(批准号:2011FY130100)资助~~
关键词
非特异性同位素稀释
激光烧蚀
电感耦合等离子体质谱
转铁蛋白
白蛋白
Species-unspecific isotope dilution
Laser ablation
Inductively coupled plasma mass spectrome-try
Transferrin
Albumin
