Max Planck Institute for Brain Research, Frankfurt, Germany.
Max Planck Institute of Biophysics, Frankfurt, Germany.
Nat Protoc. 2019 Feb;14(2):556-575. doi: 10.1038/s41596-018-0106-6.
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 ~10 d (plus the time needed to generate the mouse lines, the desired labeling period and MS analysis).
蛋白质组学的一个主要挑战是鉴定体内细胞类型特异性的蛋白质组。本方案描述了如何在活体小鼠中标记、纯化和鉴定细胞类型特异性蛋白质组。为了实现这一目标，我们创建了一个表达突变甲硫氨酰-tRNA 合成酶（L274G）的 Cre 重组酶诱导型小鼠系，该酶能够利用非典型氨基酸叠氮正亮氨酸（ANL）标记新生蛋白。这种氨基酸可以通过点击化学与不同的亲和标签结合。亲和纯化（AP）后，通过串联质谱（MS/MS）鉴定标记的蛋白质。通过这种方法，可以鉴定来自活体动物的细胞类型特异性蛋白质组，这是以前任何发表的方法都无法实现的。本方案通过减少样品复杂性，可以检测到细胞类型特异性蛋白质含量对环境变化的细微变化。这个方案大约需要 10 天（加上生成小鼠系、所需标记时间和 MS 分析所需的时间）。
