Combined tissue and fluid proteomics with Tandem Mass Tags to identify low-abundance protein biomarkers of disease in peripheral body fluid: An Alzheimer's Disease case study
Combined tissue and fluid proteomics with Tandem Mass Tags to identify low-abundance protein biomarkers of disease in peripheral body fluid: An Alzheimer's Disease case study
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DOI：
10.1002/rcm.7777
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发表时间：
2017-01-01
影响因子：
2
通讯作者：
Pike, Ian
中科院分区：
文献类型：
--
作者：
Russell, Claire L.;Heslegrave, Amanda;Pike, Ian
理想的生物标志物存在于容易获得的样品中，包括血浆和脑脊液（CSF），并且直接来自患病组织，因此可能具有相对低的丰度。由于蛋白质的高动态范围、超丰度蛋白质的优势以及数据依赖性采集质谱（MS）的使用，用于生物标志物发现的传统无偏倚蛋白质组学方法一直难以检测低丰度标志物。为了克服这些局限性，提高生物标志物的发现在外周液中，我们已经开发了TMTcalibrator;一种新的MS工作流程，结合等压标记的患病组织生物标志物平行与适当的一组标记的体液，以增加识别低丰度，组织衍生的biomarkers.MethodsA疾病相关的细胞系标记TMT（R）在一系列的浓度，产生多点校准曲线。用剩余的标签标记外周生物流体样品，并使用具有Top10 CID-HCD MS 3同步前体选择（SPS）方法的Orbitrap Fusion Tribrid质谱仪进行定量分析。SPS允许直接分析的非耗尽，未分级的CSF样品与完整的分析6个单独的样品只需要15小时的MS时间，相当于1.5小时每sample.ResultsUsing的TMT calibrator工作流程允许识别的几个标记的小胶质细胞活化的差异量化的阿尔茨海默氏病（AD）患者的CSF。我们报告的肽从41蛋白，以前没有被检测到在CSF中，这似乎是调节至少60%的AD。ConclusionsThis研究已经证明了新的TMT校准工作流程的好处，结果表明这是一个合适的和有效的方法，检测低丰度的肽内的生物液体。应考虑在进一步的生物标志物发现研究中使用TMT校准器，以克服通常与更传统方法相关的一些限制。版权所有（c）2016约翰威利父子有限公司
RationaleIdeal biomarkers are present in readily accessible samples including plasma and cerebrospinal fluid (CSF), and are directly derived from diseased tissue, therefore likely to be of relatively low abundance. Traditional unbiased proteomic approaches for biomarker discovery have struggled to detect low-abundance markers due to the high dynamic range of proteins, the predominance of hyper-abundant proteins, and the use of data-dependent acquisition mass spectrometry (MS). To overcome these limitations and improve biomarker discovery in peripheral fluids, we have developed TMTcalibrator; a novel MS workflow combining isobarically labelled diseased tissue digests in parallel with an appropriate set of labelled body fluids to increase the chance of identifying low-abundance, tissue-derived biomarkers.MethodsA disease relevant cell line was labelled with TMT (R) in a range of concentrations generating a multi-point calibration curve. Peripheral biofluid samples were labelled with the remaining tags and quantitative analysis was performed using an Orbitrap Fusion Tribrid mass spectrometer with a Top10 CID-HCD MS3 synchronous precursor selection (SPS) method. SPS allowed direct analysis of non-depleted, unfractionated CSF samples with complete profiling of six individual samples requiring only 15hours of MS time, equivalent to 1.5h per sample.ResultsUsing the TMTcalibrator workflow allowed the identification of several markers of microglia activation that are differentially quantified in the CSF of patients with Alzheimer's disease (AD). We report peptides from 41 proteins that have not previously been detected in the CSF, that appear to be regulated by at least 60% in AD.ConclusionsThis study has demonstrated the benefits of the new TMTcalibrator workflow and the results suggest this is a suitable and efficient method of detecting low-abundance peptides within biological fluids. The use of TMTcalibrator in further biomarker discovery studies should be considered to overcome some of the limitations commonly associated with more conventional approaches. Copyright (c) 2016 John Wiley & Sons, Ltd.