Cell type-specific in vivo proteomes with a multicopy mutant methionyl tRNA synthetase mouse line

The functional diversity of cells is driven by the different proteins they express. While improvements in protein labeling techniques have enabled the measurement of proteomes with increased sensitivity, measuring cell type-specific proteomes in vivo remains challenging. One of the most useful pipelines is bio-orthogonal noncanonical amino acid tagging (BONCAT) with the MetRS* system, consisting of a transgenic mouse line expressing a mutant methionyl-tRNA synthetase (MetRS*) controlled by Cre recombinase expression. This system allows cell type-specific labeling of proteins with a noncanonical amino acid (azidonorleucine, ANL), which can be subsequently conjugated to affinity or fluorescent tags using click chemistry. Click-modified proteins can then be visualized, purified and identified. The reduction in sample complexity enables the detection of small changes in protein composition. Here, we describe a multicopy MetRS* mouse line (3xMetRS* mouse line) that exhibits markedly enhanced ANL protein labeling, boosting the sensitivity and temporal resolution of the system and eliminating the need for working under methionine depletion conditions. Cell type-specific in vivo labeling is possible even in heterozygous animals, thus offering an enormous advantage for crossing the line into mutation and disease-specific backgrounds. Using the 3xMetRS* line, we identified the in vivo proteome of a sparse cell population - the dopaminergic neurons of the olfactory bulb - and furthermore determined newly synthesized proteins after short labeling durations following a single intraperitoneal ANL injection.

细胞的功能多样性由其表达的不同蛋白质所驱动。尽管蛋白质标记技术的进步已使高灵敏度检测蛋白质组成为可能，但在体内测量细胞类型特异性蛋白质组仍颇具挑战。现有最实用的技术流程之一，是结合MetRS*系统的生物正交非经典氨基酸标记（bio-orthogonal noncanonical amino acid tagging, BONCAT）：该系统由一株表达经Cre重组酶（Cre recombinase）调控的突变型甲硫氨酰-tRNA合成酶（methionyl-tRNA synthetase, MetRS*）的转基因小鼠品系构成。此系统可利用非经典氨基酸——叠氮正亮氨酸（azidonorleucine, ANL）——对细胞类型特异性蛋白质进行标记，后续可通过点击化学（click chemistry）将标记后的蛋白质与亲和标签或荧光标签相结合。经点击化学修饰的蛋白质可被可视化、纯化并鉴定。样本复杂度的降低使得检测蛋白质组成的微小变化成为可能。本文介绍了一株多拷贝MetRS*小鼠品系（3xMetRS*小鼠品系），该品系的ANL蛋白质标记效率显著提升，增强了该系统的灵敏度与时间分辨率，且无需在甲硫氨酸缺失条件下开展实验。即便在杂合子动物中，也可实现细胞类型特异性的体内标记，这为将该品系与突变及疾病特异性背景品系杂交提供了极大优势。利用3xMetRS*小鼠品系，我们成功鉴定了稀疏细胞群体——嗅球多巴胺能神经元——的体内蛋白质组，此外还在单次腹腔注射ANL后，通过短时长标记确定了新合成的蛋白质。