Dowling Paul, Ohlendieck Kay
Department of Biology, Maynooth University, National University of Ireland, Maynooth, Co. Kildare, Ireland.
Methods Mol Biol. 2018;1664:245-257. doi: 10.1007/978-1-4939-7268-5_19.
This chapter will focus on upstream immunodepletion of high abundant proteins from plasma samples and subsequent analysis by difference gel electrophoresis (DIGE). The abundances of proteins in biofluid proteomes, such as serum, plasma, saliva, and bronchoalveolar lavage fluid (BALF), can exceed 10 orders of magnitude. This substantial dynamic range is problematic for the detection of medium and low abundance proteins by DIGE analysis. To increase the detection, quantification, and identification of medium-low abundant proteins, the targeted depletion of known abundant proteins with antibody columns has been successfully employed. From the literature, it is clear that the performance of abundant protein depletion with immunodepletion columns has been successful in broadening the coverage of the biofluid proteome and facilitating the identification of disease-specific biomarkers. The task for a successful biomarker strategy involves the combination of a reproducible and robust fractionation method, coupled with a highly accurate quantitative method, a task that is exemplified by combining both immunodepletion and DIGE together to discover significant proteins associated with the disease phenotype.
本章将重点介绍从血浆样本中对高丰度蛋白质进行上游免疫去除，以及随后通过差异凝胶电泳（DIGE）进行分析。生物流体蛋白质组（如血清、血浆、唾液和支气管肺泡灌洗液（BALF））中蛋白质的丰度可超过10个数量级。如此大的动态范围对于通过DIGE分析检测中低丰度蛋白质而言是个问题。为了增加对中低丰度蛋白质的检测、定量和鉴定，已成功采用抗体柱对已知的高丰度蛋白质进行靶向去除。从文献中可以清楚地看到，使用免疫去除柱去除高丰度蛋白质的性能已成功拓宽了生物流体蛋白质组的覆盖范围，并有助于鉴定疾病特异性生物标志物。成功的生物标志物策略的任务涉及将可重复且稳健的分级分离方法与高度准确的定量方法相结合，将免疫去除和DIGE结合在一起以发现与疾病表型相关的重要蛋白质就是这一任务的例证。
