Dual-immuno-MS technique for improved differentiation power in heterodimeric protein biomarker analysis: determination and differentiation of human chorionic gonadotropin variants in serum

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• 作者：Siri Valen Egeland ; Léon Reubsaet ; Elisabeth Paus…
• 关键词：Immunocapture ; LC ; MS/MS ; Differentiation power ; Proteins ; Biomarkers ; Heterodimers
• 刊名：Analytical and Bioanalytical Chemistry
• 出版年：2016
• 出版时间：October 2016
• 年：2016
• 卷：408
• 期：26
• 页码：7379-7391
• 全文大小：1,063 KB
• 刊物类别：Chemistry and Materials Science
• 刊物主题：Chemistry
  Analytical Chemistry
  Food Science
  Inorganic Chemistry
  Physical Chemistry
  Monitoring, Environmental Analysis and Environmental Ecotoxicology
  
• 出版者：Springer Berlin / Heidelberg
• ISSN：1618-2650
• 卷排序：408

文摘

If the biomarker potential of intact heteromers and their free subunits is different, differentiation between these forms may reveal important clinical information. Such differentiation may however be analytically challenging. One possible way of circumventing this challenge is by performing a dual-immuno-MS approach. In the present paper, a two-step immunoaffinity sample preparation step is succeeded by digestion and subsequent LC-MS analysis to provide high-sensitivity quantification and differentiation between the heterodimer human chorionic gonadotropin (hCG) and its free β-subunit in serum. Intact and free variants are captured in two separate immunoextraction steps in order to increase the differentiation power of the method. Intact heterodimer variants were depleted prior to free subunit variants in order to incorporate a method quality control. The method was optimized for serum samples. A fully validated immuno-MS method was used as foundation, and partial validation according to the European Medicines Agency’s (EMA) guidelines on validation of bioanalytical methods was performed for the dual approach. An accelerated digestion step was incorporated making batch processing of samples within 1 day possible (approx. 3.5 h of sample preparation including digestion). Acceptable linearity (R2 ≥ 0.990 for four variants and R2 of 0.920 and 0.966 for the remaining two) and specificity were demonstrated, and the method was robust toward varying levels of intact heterodimer versus free subunit. The method was also successfully tested on realistic samples, demonstrating both the differences in total hCG and the distribution between intact hCG and its free β-subunit in real samples.