The Effect of Pre-Analytical Variability on the Measurement of MRM-MS-Based Mid- to High-Abundance Plasma Protein Biomarkers and a Panel of Cytokines
The Effect of Pre-Analytical Variability on the Measurement of MRM-MS-Based Mid- to High-Abundance Plasma Protein Biomarkers and a Panel of Cytokines
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DOI：
10.1371/journal.pone.0038290
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发表时间：
2012-06-06
期刊：
影响因子：
3.7
通讯作者：
Basik, Mark
中科院分区：
文献类型：
--
作者：
Aguilar-Mahecha, Adriana;Kuzyk, Michael A.;Basik, Mark
血液样本的处理和操作可能对生物标志物研究中测量的蛋白质的稳定性和水平产生显著影响。需要在可用于生物标志物发现和验证的不同蛋白质组学平台的背景下很好地理解这种分析前的可变性。在本研究中，我们评估了不同类型的采血管，包括含有蛋白酶抑制剂的BD P100采血管以及可防止血小板活化的Cyclop采血管。我们研究了不同处理方案以及试管处理延迟对使用新型多反应监测-质谱（MRM-MS）测定的55种中高丰度血浆蛋白水平以及使用市售多重珠基免疫测定的27种低丰度细胞因子水平的影响。使用含有蛋白酶抑制剂的P100管仅赋予4种细胞因子和仅一种MRM-MS测量的肽的蛋白水解保护。通过MRM测量的中丰度和高丰度蛋白质在未经处理的血浆中高度稳定长达6小时，尽管血小板活化也会影响这些蛋白质的水平。当试管在低温下离心时，细胞因子的水平升高，而当样品收集在离心管中时，检测到低水平的细胞因子。根据所用收集管的类型，离心延迟也对测量的细胞因子水平产生影响。我们的研究结果可以帮助制定蛋白质组生物标志物研究的血液采集和处理指南。
Blood sample processing and handling can have a significant impact on the stability and levels of proteins measured in biomarker studies. Such pre-analytical variability needs to be well understood in the context of the different proteomics platforms available for biomarker discovery and validation. In the present study we evaluated different types of blood collection tubes including the BD P100 tube containing protease inhibitors as well as CTAD tubes, which prevent platelet activation. We studied the effect of different processing protocols as well as delays in tube processing on the levels of 55 mid and high abundance plasma proteins using novel multiple-reaction monitoring-mass spectrometry (MRM-MS) assays as well as 27 low abundance cytokines using a commercially available multiplexed bead-based immunoassay. The use of P100 tubes containing protease inhibitors only conferred proteolytic protection for 4 cytokines and only one MRM-MS-measured peptide. Mid and high abundance proteins measured by MRM are highly stable in plasma left unprocessed for up to six hours although platelet activation can also impact the levels of these proteins. The levels of cytokines were elevated when tubes were centrifuged at cold temperature, while low levels were detected when samples were collected in CTAD tubes. Delays in centrifugation also had an impact on the levels of cytokines measured depending on the type of collection tube used. Our findings can help in the development of guidelines for blood collection and processing for proteomic biomarker studies.