Synthesis and evaluation of a novel adenosine-ribose probe for global-scale profiling of nucleoside and nucleotide-binding proteins.
Synthesis and evaluation of a novel adenosine-ribose probe for global-scale profiling of nucleoside and nucleotide-binding proteins.
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DOI：
10.1371/journal.pone.0115644
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发表时间：
2015
期刊：
影响因子：
3.7
通讯作者：
Stevens SM Jr
中科院分区：
文献类型：
--
作者：
Mahajan S;Manetsch R;Merkler DJ;Stevens SM Jr
蛋白质组学是研究疾病发生和发展的复杂分子机制的有力手段。现代蛋白质谱分析方法中的一个重要挑战涉及靶向特定蛋白质活性以鉴定与疾病病理生理学相关的改变的分子过程。腺苷结合蛋白是蛋白质组的一个重要亚类，其异常表达或活性变化与多种人类疾病有关。在此，我们描述了一种基于亲和力的方法，用于从复杂的细胞蛋白质组中富集腺苷结合蛋白。合成了一种新型的N6-生物素化的8-叠氮基-腺苷探针（NXR探针），它含有一个与靶蛋白形成共价键的反应基团，以及一个生物素标签，用于亲和素层析的亲和富集。在由源自小鼠神经母细胞瘤N18 TG 2细胞的裂解物组成的复杂蛋白质混合物中进一步评价之前，用蛋白质标准品确认探针特异性。蛋白质鉴定和相对定量使用质谱法允许在这些样品中的核苷和核苷酸结合蛋白的丰度的小的变化，在标记的蛋白质组从神经母细胞瘤细胞中观察到一个显着富集的miR-结合蛋白的鉴定。这项研究的结果表明，这种方法可以在复杂的蛋白质混合物中富集核苷和核苷酸结合蛋白，指向一组独特的蛋白质，可以在进一步了解疾病机制或基本生物过程的背景下进行检查。
Proteomics is a powerful approach used for investigating the complex molecular mechanisms of disease pathogenesis and progression. An important challenge in modern protein profiling approaches involves targeting of specific protein activities in order to identify altered molecular processes associated with disease pathophysiology. Adenosine-binding proteins represent an important subset of the proteome where aberrant expression or activity changes of these proteins have been implicated in numerous human diseases. Herein, we describe an affinity-based approach for the enrichment of adenosine-binding proteins from a complex cell proteome. A novel N 6-biotinylated-8-azido-adenosine probe (AdoR probe) was synthesized, which contains a reactive group that forms a covalent bond with the target proteins, as well as a biotin tag for affinity enrichment using avidin chromatography. Probe specificity was confirmed with protein standards prior to further evaluation in a complex protein mixture consisting of a lysate derived from mouse neuroblastoma N18TG2 cells. Protein identification and relative quantitation using mass spectrometry allowed for the identification of small variations in abundance of nucleoside- and nucleotide-binding proteins in these samples where a significant enrichment of AdoR-binding proteins in the labeled proteome from the neuroblastoma cells was observed. The results from this study demonstrate the utility of this method to enrich for nucleoside- and nucleotide-binding proteins in a complex protein mixture, pointing towards a unique set of proteins that can be examined in the context of further understanding mechanisms of disease, or fundamental biological processes in general.
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DOI：
10.1038/newbio235225a0
发表时间：
1972-01-01
期刊：
NATURE-NEW BIOLOGY
影响因子：
--
作者：
MINNA, J;NIRENBERG, M;GLAZER, D
通讯作者：
GLAZER, D
影响因子：
7.3
作者：
Roelen, H;Veldman, N;IJzerman, AP
通讯作者：
IJzerman, AP
影响因子：
4
作者：
Dong, Su-Wei;Wang, Lin;Song, Xin
通讯作者：
Song, Xin
影响因子：
2.7
作者：
NAGATA, K;SUZUKI, K;IMAHORI, K
通讯作者：
IMAHORI, K
影响因子：
2.9
作者：
POMERANTZ, AH;RUDOLPH, SA;GREENGARD, P
通讯作者：
GREENGARD, P