Rapid label-free identification of estrogen-induced differential protein expression in vivo from mouse brain and uterine tissue.
Rapid label-free identification of estrogen-induced differential protein expression in vivo from mouse brain and uterine tissue.
复制标题
DOI：
10.1021/pr900083v
复制
发表时间：
2009-08
影响因子：
4.4
通讯作者：
Nguyen V
中科院分区：
文献类型：
--
作者：
Prokai L;Stevens SM Jr;Rauniyar N;Nguyen V
采用液相色谱-串联质谱法为基础的“鸟枪法”蛋白质组学和无标记相对定量的组织蛋白质丰度分布进行了评价，用于研究小鼠大脑和子宫中雌激素调节的蛋白质表达。样品制备涉及在8 M尿素水溶液中进行30分钟的蛋白质提取，然后对提取的蛋白质进行二硫化物还原、硫醇烷基化和胰蛋白酶消化，并在3-4 mg组织上进行，以评价该方法在动物模型中加速受实验性治疗干预体内影响的细胞途径的调查的适用性。无标记蛋白质组学方法（光谱计数）适用于鉴定皮质蛋白质水平的细微变化，并显示雌激素诱导的ATP合酶（α和β亚型）、天冬氨酸转氨酶2和线粒体苹果酸脱氢酶显著上调，而无需对组织进行任何预先亚细胞分离或使用多维色谱分离。该方法也适用于观察17β-雌二醇治疗后卵巢切除小鼠子宫组织中各种上调和下调的蛋白质。除了确认谷胱甘肽S-转移酶的丰度非常显着减少，被认为是雌激素的影响的标志物，我们的研究还揭示了潜在的新的蛋白质标志物，如结蛋白和lumican，细胞骨架排列的关键组成部分，因此，调节其丰度可能有助于雌激素刺激后发生的子宫主要形态学变化。
Protein abundance profiling from tissue using liquid chromatograph—tandem mass spectrometry-based ‘shotgun’ proteomics and label-free relative quantitation was evaluated for the investigation of estrogen-regulated protein expression in the mouse brain and uterus. Sample preparation involved a 30-min protein extraction in 8 M aqueous urea solution, followed by disulphide reduction, thiol alkylation and trypsin digestion of the extracted proteins, and was performed on 3–4 mg of tissue in order to evaluate the suitability of this methodology to expedite the survey of cellular pathways that are affected in vivo by an experimental therapeutic intervention in an animal model. The label-free proteomic approach (spectral counting) was suitable to identify even subtle changes in cortical protein levels and revealed significant estrogen-induced upregulation of ATP synthase (both α- and β-isoforms), aspartate aminotransferase 2 and mitochondrial malate dehydrogenase without any prior subcellular fractionation of the tissue or the use of multidimensional chromatographic separation. The methodology was also suitable to observe various up- and downregulated proteins in the uterine tissue of ovariectomized mice upon treatment with 17β-estradiol. In addition to confirming a very significant decrease in the abundance of glutathione S-transferase recognized as a marker of estrogen’s impact, our studies have also revealed potential new protein markers such as desmin and lumican that are critical components of cytoskeletal arrangement and, hence, regulation of their abundance could contribute to major morphological changes in the uterus occurring upon estrogenic stimulation.
登录
查看更多内容
影响因子：
5.3
作者：
Nilsen, Jon;Irwin, Ronald W.;Brinton, Roberta Diaz
通讯作者：
Brinton, Roberta Diaz
影响因子：
2.9
作者：
Xixi, E;Dimitraki, P;Fountoulakis, M
通讯作者：
Fountoulakis, M
DOI：
10.1111/j.2517-6161.1995.tb02031.x
发表时间：
1995-01-01
影响因子：
5.8
作者：
BENJAMINI, Y;HOCHBERG, Y
通讯作者：
HOCHBERG, Y
影响因子：
7.4
作者：
Liu, HB;Sadygov, RG;Yates, JR
通讯作者：
Yates, JR
影响因子：
2.9
作者：
Krapfenbauer, K;Fountoulakis, M;Lubec, G
通讯作者：
Lubec, G