Cell-type-specific metabolic labeling, detection and identification of nascent proteomes in vivo
Cell-type-specific metabolic labeling, detection and identification of nascent proteomes in vivo
复制标题
DOI：
10.1038/s41596-018-0106-6
复制
发表时间：
2019-02-01
期刊：
影响因子：
14.8
通讯作者：
Schuman, Erin M.
中科院分区：
文献类型：
--
作者：
Alvarez-Castelao, Beatriz;Schanzenbaecher, Christoph T.;Schuman, Erin M.
蛋白质组学的一大挑战是在体内鉴定细胞类型特异性蛋白质组。该方案描述了如何标记，纯化和鉴定活小鼠中的细胞类型特异性蛋白质组。为了使这成为可能，我们创建了一个Cre重组酶诱导型小鼠系，表达突变型甲硫氨酰-tRNA合成酶（L274 G），这使得新生蛋白质的标记与非规范氨基酸叠氮正亮氨酸（ANL）。该氨基酸可以通过点击化学与不同的亲和标签缀合。在亲和纯化（AP）后，标记的蛋白质可以通过串联质谱（MS/MS）鉴定。通过这种方法，可以鉴定来自活动物的细胞类型特异性蛋白质组，这是任何以前发表的方法都不可能的。通过该方案实现的样品复杂性的降低允许检测响应于环境变化的细胞类型特异性蛋白质含量的细微变化。该方案可以在类似于10天内完成（加上产生小鼠品系所需的时间、所需的标记期和MS分析）。
A big challenge in proteomics is the identification of cell-type-specific proteomes in vivo. This protocol describes how to label, purify and identify cell-type-specific proteomes in living mice. To make this possible, we created a Cre-recombinase-inducible mouse line expressing a mutant methionyl-tRNA synthetase (L274G), which enables the labeling of nascent proteins with the non-canonical amino acid azidonorleucine (ANL). This amino acid can be conjugated to different affinity tags by click chemistry. After affinity purification (AP), the labeled proteins can be identified by tandem mass spectrometry (MS/MS). With this method, it is possible to identify cell-type-specific proteomes derived from living animals, which was not possible with any previously published method. The reduction in sample complexity achieved by this protocol allows for the detection of subtle changes in cell-type-specific protein content in response to environmental changes. This protocol can be completed in similar to 10 d (plus the time needed to generate the mouse lines, the desired labeling period and MS analysis).