Two-color labeling of temporally defined protein populations in mammalian cells.
Two-color labeling of temporally defined protein populations in mammalian cells.
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DOI：
10.1016/j.bmcl.2008.08.046
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发表时间：
2008-11-15
影响因子：
2.7
通讯作者：
Tirrell DA
中科院分区：
文献类型：
--
作者：
Beatty KE;Tirrell DA
蛋白质组在疾病、药物治疗和正常细胞信号传导过程中发生复杂的变化。这种变化的表征需要时间分辨蛋白质鉴定和成像的方法。在这里，我们描述了两个反应性甲硫氨酸（Met）类似物，叠氮基高丙氨酸（Aha）和高炔丙基甘氨酸（Hpg），在固定的细胞中标记两个蛋白质群体的应用。活性丽丝胺罗丹明（LR），7-二甲基氨基香豆素（DMAC），和Bodipy-630（BDPY）染料的制备和检查用于选择性染料标记的新合成的蛋白质在大鼠-1成纤维细胞。LR和DMAC，但不是BDPY，荧光团被发现能够选择性地，有效地标记蛋白质组的子集;用Aha和Hpg标记的细胞表现出比用Met处理的对照细胞强3至7倍的荧光发射。我们还研究了同时和顺序脉冲标记的细胞与AHA和HPG。脉冲标记后，用反应性LR和DMAC染料处理细胞，并通过荧光显微镜对标记的细胞进行成像，并通过流式细胞术进行分析。这些研究的结果表明，氨基酸标记可用于实现哺乳动物细胞中的时间定义的蛋白质群体的选择性双色成像。
The proteome undergoes complex changes in response to disease, drug treatment, and normal cellular signaling processes. Characterization of such changes requires methods for time-resolved protein identification and imaging. Here we describe the application of two reactive methionine (Met) analogues, azidohomoalanine (Aha) and homopropargylglycine (Hpg), to label two protein populations in fixed cells. Reactive lissamine rhodamine (LR), 7-dimethylaminocoumarin (DMAC), and Bodipy-630 (BDPY) dyes were prepared and examined for use in selective dye-labeling of newly synthesized proteins in Rat-1 fibroblasts. The LR and DMAC, but not BDPY, fluorophores were found to enable selective, efficient labeling of subsets of the proteome; cells labeled with Aha and Hpg exhibited fluorescence emission 3- to 7-fold more intense than that of control cells treated with Met. We also examined simultaneous and sequential pulse-labeling of cells with Aha and Hpg. After pulse-labeling, cells were treated with reactive LR and DMAC dyes, and labeled cells were imaged by fluorescence microscopy and analyzed by flow cytometry. The results of these studies demonstrate that amino acid labeling can be used to achieve selective two-color imaging of temporally defined protein populations in mammalian cells.
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影响因子：
3.5
作者：
Kiick, KL;Weberskirch, R;Tirrell, DA
通讯作者：
Tirrell, DA
DOI：
10.1083/jcb.200211046
发表时间：
2003-01-20
期刊：
The Journal of cell biology
影响因子：
--
作者：
Chen H;Detmer SA;Ewald AJ;Griffin EE;Fraser SE;Chan DC
通讯作者：
Chan DC
DOI：
10.1073/pnas.0506855103
发表时间：
2006-03-28
影响因子：
11.1
作者：
Dube, DH;Prescher, JA;Bertozzi, CR
通讯作者：
Bertozzi, CR
DOI：
10.1073/pnas.0601637103
发表时间：
2006-06-20
影响因子：
11.1
作者：
Dieterich, Daniela C.;Link, A. James;Schuman, Erin M.
通讯作者：
Schuman, Erin M.
影响因子：
15
作者：
Agard, NJ;Prescher, JA;Bertozzi, CR
通讯作者：
Bertozzi, CR