Efficient Molecular Imprinting Strategy for Quantitative Targeted Proteomics of Human Transferrin Receptor in Depleted Human Serum.
Efficient Molecular Imprinting Strategy for Quantitative Targeted Proteomics of Human Transferrin Receptor in Depleted Human Serum.
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DOI：
10.1021/acs.analchem.5b02633
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发表时间：
2015-10
影响因子：
7.4
通讯作者：
Liang Liu;Ting Zhong;Qingqing Xu;Yun Chen
中科院分区：
文献类型：
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作者：
Liang Liu;Ting Zhong;Qingqing Xu;Yun Chen
血清可溶性转铁蛋白受体(STfR)已被认为是乳腺癌诊断、监测和治疗的标志物。然而，在某些情况下，sTfR水平可能远远低于大多数检测的定量限度(LOQ)。因此，需要一种有效的样品前处理策略。在本研究中，我们开发了分子印迹聚合物(MIP)，并将其与基于LC-MS/MS的靶向蛋白质组学相结合用于sTfR的检测。这项工作的关键是相同的sTfR代理肽(VEYHFLSPYVSPK，VK13)被用于MIP的富集物和靶向蛋白质组学的定量。具体地说，考察了三种不同长度的多肽模板对分子印迹聚合物合成的影响，并优化了印迹条件。研究了分子印迹聚合物的吸附容量、吸附动力学和结合选择性等特性。结果，使用分子印迹聚合物，∼的灵敏度提高了12倍。最低检测浓度为200 ng·mL(-1)。日内精密度为10.7%，日间精密度为7.8%。定量下限(LLOQ)的准确度为7.5%，其他质控水平的准确度为8.4%。经验证后，用标准加入法测定乳腺癌患者(n=20)和健康志愿者(n=20)的sTfR水平。受试者血清sTfR水平为1.5 9±0.36μg·m L(-1)(范围0.96~2.34μg·m L-1)和1.82±0.42μg·m L(范围0.95~2.4 7μg.这项研究是首次将分子印迹聚合物与LC-MS/MS靶向蛋白质组学相结合，在多肽水平上进行蛋白质定量。
Soluble transferrin receptor (sTfR) in serum has been suggested as a marker for breast cancer diagnosis, monitoring and treatment. However, sTfR levels in some situations could be far below the limit of quantification (LOQ) of most assays. Thus, an efficient sample pretreatment strategy is required. In this study, molecularly imprinted polymers (MIPs) were developed and coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based targeted proteomics for sTfR measurement. The key to this effort was that the same surrogate peptide of sTfR (VEYHFLSPYVSPK, VK13) was employed in both the enrichment by MIPs and the quantification by targeted proteomics. Specifically, three peptide templates with different lengths were evaluated for the synthesis of MIPs, and the imprinting conditions were optimized. The characteristics of MIPs, including the adsorption capacity, adsorption kinetics, and binding selectivity, were also investigated. As a result, a ∼12-fold enhancement of sensitivity was achieved using MIPs. An LOQ of 200 ng·mL(-1) was obtained. The intra- and interday precision were <10.7 and 7.8%, respectively. The accuracy was 7.5% at the lower limit of quantification (LLOQ) and <8.4% for the other QC levels. After validation, the assay was applied to determine the sTfR levels in breast cancer patients (n = 20) and healthy volunteers (n = 20) using the standard addition method. The corresponding levels of sTfR were 1.59 ± 0.36 μg·mL(-1) (range: 0.96-2.34 μg·mL(-1)) in the volunteers and 1.82 ± 0.42 μg·mL(-1) (range: 0.95-2.47 μg·mL(-1)) in the patients. This study is among the first to combine MIPs and LC-MS/MS targeted proteomics for protein quantification at the peptide level.