Max-Planck-Institute for Brain Research, Synaptic Plasticity Department.
Department of Biochemistry and Molecular Biology, Veterinary School, Complutense University of Madrid.
J Vis Exp. 2022 Apr 13(182). doi: 10.3791/63713.
Understanding protein homeostasis in vivo is key to knowing how the cells work in both physiological and disease conditions. The present protocol describes in vivo labeling and subsequent purification of newly synthesized proteins using an engineered mouse line to direct protein labeling to specific cellular populations. It is an inducible line by Cre recombinase expression of L274G-Methionine tRNA synthetase (MetRS*), enabling azidonorleucine (ANL) incorporation to the proteins, which otherwise will not occur. Using the method described here, it is possible to purify cell-type-specific proteomes labeled in vivo and detect subtle changes in protein content due to sample complexity reduction.
了解体内蛋白质的动态平衡对于了解细胞在生理和疾病状态下的工作方式至关重要。本方案描述了使用工程化的小鼠品系对新合成的蛋白质进行体内标记和随后的纯化，以将蛋白质标记导向特定的细胞群体。这是一种通过 Cre 重组酶表达 L274G-蛋氨酸 tRNA 合成酶（MetRS*）诱导的品系，使氮杂正亮氨酸（ANL）掺入蛋白质中，否则蛋白质不会掺入。使用这里描述的方法，可以纯化体内标记的细胞类型特异性蛋白质组，并检测由于样品复杂性降低而导致的蛋白质含量的细微变化。
