A new liquid chromatography-mass spectrometry-based method to quantitate exogenous recombinant transferrin in cerebrospinal fluid: a potential approach for pharmacokinetic studies of transferrin-based therapeutics in the central nervous systems.
A new liquid chromatography-mass spectrometry-based method to quantitate exogenous recombinant transferrin in cerebrospinal fluid: a potential approach for pharmacokinetic studies of transferrin-based therapeutics in the central nervous systems.
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DOI：
10.1255/ejms.1365
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发表时间：
2015
期刊：
影响因子：
--
通讯作者：
Kaltashov IA
中科院分区：
文献类型：
--
作者：
Wang S;Bobst CE;Kaltashov IA
转铁蛋白（Tf）是一种80 kDa的铁结合蛋白，由于其能够穿透血脑屏障（BBB），被认为是一种有前途的靶向中枢神经系统的药物载体。在开发以Tf为基础的治疗方法的诸多挑战中，由于大量内源性Tf的存在，对脑脊液（CSF）中给药Tf的敏感和准确定量仍然特别困难。在此，我们描述了一种新的基于LC-MS的方法，用于灵敏和准确地定量大鼠脑脊液中外源性重组人Tf。通过利用重组Tf中存在的His-tag并应用Ni亲和纯化，尽管存在丰富的内源蛋白，但外源hTf可以从大鼠脑脊液中大量富集。此外，我们应用了新开发的o18标记技术，该技术可以在蛋白质水平上生成内部标准，大大提高了定量的准确性和稳健性。对所建立的方法进行了线性度、准确度、精密度和定量下限的考察，均符合生物分析方法验证的普遍标准。
Transferrin (Tf) is an 80 kDa iron-binding protein which is viewed as a promising drug carrier to target the central nervous system due to its ability to penetrate the blood-brain barrier (BBB). Among the many challenges during the development of Tf-based therapeutics, sensitive and accurate quantitation of the administered Tf in cerebrospinal fluid (CSF) remains particularly difficult due to the presence of abundant endogenous Tf. Herein, we describe the development of a new LC-MS based method for sensitive and accurate quantitation of exogenous recombinant human Tf in rat CSF. By taking advantage of a His-tag present in recombinant Tf and applying Ni affinity purification, the exogenous hTf can be greatly enriched from rat CSF, despite the presence of the abundant endogenous protein. Additionally, we applied a newly developed O18-labeling technique that can generate internal standards at the protein level, which greatly improved the accuracy and robustness of quantitation. The developed method was investigated for linearity, accuracy, precision and lower limit of quantitation, all of which met the commonly accepted criteria for bioanalytical method validation.
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