A Novel Platform for Maternal Alcohol Consumption Screening

描述（由申请人提供）：孕妇在怀孕期间滥用酒精可导致胎儿酒精谱系障碍（FASD），这是一种以一系列神经解剖和神经行为缺陷为特征的终身残疾。目前估计FASD在美国学龄儿童中的患病率约为2-5%。因此，早期发现孕妇饮酒对预防FASD至关重要。产妇自我报告在很大程度上是不可靠的，目前的生物标志物的效用在怀孕期间也受到限制。在这些标志物中，转铁蛋白的糖基化变体（碳水化合物缺乏转铁蛋白，CDT）被报道为慢性酒精滥用的最具诊断特异性的生物标志物。然而，根据所采用的分析方法，平均灵敏度约为~65.4%，范围很广。此外，利用传统的电泳、色谱和免疫方法，迄今为止还难以表征所有糖型的组成和丰度。这是至关重要的，因为酒精会对聚糖产生复杂的影响，包括唾液酸的损失、糖的部分损失和/或低聚糖链的完全损失。在一个广泛应用的FASD大鼠模型中，我们首次使用了最先进的质谱技术，并获得了初步数据，表征了母体血浆转铁蛋白N-连接寡糖的序列和丰度，并保留了N-糖苷的位置。基于我们的新数据，我们提出以下具体目标：在FASD大鼠模型中，利用传统的生物标志物（转铁蛋白变体）开发并验证一种新的、敏感的高通量平台，用于筛选母体酒精消耗。研究人员提出了两个子目标，以表征和验证母体血浆中蛋白质翻译后糖基化特征丰度，并将其与酒精暴露的剂量和持续时间联系起来。我们将利用怀孕大鼠模型暴露于分级酒精剂量在妊娠期间给予不同的暴露时间。血浆蛋白（转铁蛋白）鉴定，序列定位，糖肽鉴定与诊断片段谱，高分辨率/准确的质量定量将进行。验证研究将包括n-连接的糖苷确认和基于凝集素的探测。基于我们的初步数据，我们期望所提出的平台提供一个高度敏感的蛋白质翻译后修饰特征谱，这将大大增加检测窗口

DESCRIPTION (provided by applicant): Maternal alcohol abuse during pregnancy can result in Fetal Alcohol Spectrum Disorders (FASD), a lifelong disability characterized by a range of neuroanatomical and neurobehavioral deficits. Current estimates of FASD prevalence is about 2-5% among young school children in the United States. Therefore, early detection of alcohol use among pregnant women is critical to prevent FASD. Maternal self-reporting is largely unreliable and the utility of current biomarkers is also limited during pregnancy. Among these markers, glycosylated variants of the protein transferrin (carbohydrate deficient transferrin, CDT), is reported to be the most diagnostically specific biomarker for chronic alcohol abuse. However, the mean sensitivity is about ~65.4% with a wide range depending upon the analytical method employed. Moreover, utilizing the conventional electrophoretic, chromatographic, and immunometric methods, it has so far been difficult to characterize the composition and abundance of all glycoforms. This is critical because alcohol produces complex effects on the glycans including loss of sialic acids, partial loss of sugars, and/or complete loss of oligosaccharide chains. In a widely utilized FASD rat model, we have for the first time utilized state of the art mass spectrometry and obtained preliminary data characterizing the sequences and abundances of maternal plasma transferrin N- linked oligosaccharides preserving the location of the N-glycosite. Based on our novel data, we propose the following Specific Aim: Develop and validate a novel and sensitive high throughput platform utilizing a traditional biomarker (variants of transferrin) for maternal alcohol consumption screening in a FASD rat model. Two sub aims are proposed to characterize and validate the protein's post-translational glycosylation signature abundance profile in the maternal plasma and relating it to the dose and duration of alcohol exposure. We will utilize a pregnant rat model exposed to graded alcohol doses administered for different durations of exposure during gestation. Plasma protein (transferrin) identification, sequence mapping, glycopeptide identification with diagnostic fragmentation spectra, and High Resolution/Accurate Mass quantitation will be performed. Validation studies will include N-linked Glycosite confirmation and lectin-based probing. Based on our preliminary data, we expect the proposed platform to provide a highly sensitive protein post-translational modification signature profile that will greatly enhance the window of detection and also clearly distinguish between the alcohol doses. At the end, we plan to correlate the glycosylation signature profile with FASD neurobehavioral measures. We also believe that these novel methodologies can be in the future extended to pregnant women and form the basis for a high throughput screening tool that will help establish a standardized diagnostic criteria with a real clinical impact not only in diagnosis but also in treatment and prevention.