Department of Biology, Maynooth University, National University of Ireland, Maynooth, Co. Kildare, Ireland.
Methods Mol Biol. 2023;2596:363-375. doi: 10.1007/978-1-0716-2831-7_25.
This chapter focuses on upstream immunodepletion of high-abundance proteins from plasma samples and subsequent analysis by fluorescence two-dimensional difference gel electrophoresis (2D-DIGE). The abundances of proteins in biofluid proteomes, such as serum, plasma, saliva, and bronchoalveolar lavage fluid (BALF), can exceed ten orders of magnitude. This substantial dynamic range is problematic for the detection of medium and low-abundance proteins by 2D-DIGE analysis. To increase the detection, quantification, and identification of medium-low-abundance 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 2D-DIGE together to discover significant proteins associated with the disease phenotype.
本章重点介绍从血浆样品中进行上游高丰度蛋白免疫耗竭，然后通过荧光二维差异凝胶电泳（2D-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））中的蛋白质丰度可以超过十个数量级。这种巨大的动态范围对于通过 2D-DIGE 分析检测中低丰度蛋白质是一个问题。为了增加中低丰度蛋白质的检测、定量和鉴定，已经成功地使用抗体柱对已知的丰富蛋白质进行靶向耗竭。从文献中可以清楚地看出，免疫耗竭柱对丰富蛋白质的耗竭性能已成功地拓宽了生物流体蛋白质组的覆盖范围，并促进了疾病特异性生物标志物的鉴定。成功的生物标志物策略的任务涉及到将可重复和稳健的分级方法与高度准确的定量方法相结合，这一任务的一个例子是将免疫耗竭和 2D-DIGE 结合起来，以发现与疾病表型相关的显著蛋白质。
