Multiple Reaction Monitoring Enables Precise Quantification of 97 Proteins in Dried Blood Spots
Multiple Reaction Monitoring Enables Precise Quantification of 97 Proteins in Dried Blood Spots
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DOI：
10.1074/mcp.o115.049957
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发表时间：
2015-11-01
影响因子：
7
通讯作者：
Borchers, Christoph H.
中科院分区：
文献类型：
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作者：
Chambers, Andrew G.;Percy, Andrew J.;Borchers, Christoph H.
干血斑（DBS）方法提供了一种微创的样本采集方法，并使大多数分析物能够在室温下储存。DBS样品已成功地通过液相色谱多反应监测质谱（LC/MRM-MS）进行分析，以量化大范围的小分子生物标志物和药物;然而，这种策略最近才被探索用于基于MS的蛋白质组学应用。在这里，我们报告了一个高度多路复用的MRM测定，以定量内源性蛋白质在人类DBS样本的发展。该测定使用匹配的稳定同位素标记的标准肽进行精确的相对定量，并使用标准曲线表征分析性能。在最终试验中，共定量了169种肽，对应于97种蛋白质，平均线性动态范围为207倍，平均R-2值为0.987。该测定的总范围跨越了近5个数量级，从18.0 mg/ml的血清白蛋白（P02768）到190 ng/ml的胆碱酯酶（P06276）。6份生物样品的平均批内和批间精密度范围分别为6.1 - 7.5%CV和9.5 - 11.0%CV。大多数肽靶标在-20 ℃至37 ℃的储存温度下储存154天后是稳定的。此外，匹配的DBS和全血样本之间的蛋白质浓度比在很大程度上是恒定的（
The dried blood spot (DBS) methodology provides a minimally invasive approach to sample collection and enables room-temperature storage for most analytes. DBS samples have successfully been analyzed by liquid chromatography multiple reaction monitoring mass spectrometry (LC/MRM-MS) to quantify a large range of small molecule biomarkers and drugs; however, this strategy has only recently been explored for MS-based proteomics applications. Here we report the development of a highly multiplexed MRM assay to quantify endogenous proteins in human DBS samples. This assay uses matching stable isotope-labeled standard peptides for precise, relative quantification, and standard curves to characterize the analytical performance. A total of 169 peptides, corresponding to 97 proteins, were quantified in the final assay with an average linear dynamic range of 207-fold and an average R-2 value of 0.987. The total range of this assay spanned almost 5 orders of magnitude from serum albumin (P02768) at 18.0 mg/ml down to cholinesterase (P06276) at 190 ng/ml. The average intra-assay and inter-assay precision for 6 biological samples ranged from 6.1-7.5% CV and 9.5-11.0% CV, respectively. The majority of peptide targets were stable after 154 days at storage temperatures from -20 degrees C to 37 degrees C. Furthermore, protein concentration ratios between matching DBS and whole blood samples were largely constant (