Center for Tissue Regeneration, Repair and Restoration , V.A. Palo Alto Healthcare System , Palo Alto , California 94304 , United States.
J Am Chem Soc. 2018 Jun 13;140(23):7046-7051. doi: 10.1021/jacs.8b03074. Epub 2018 May 30.
Bioorthogonal tools enable cell-type-specific proteomics, a prerequisite to understanding biological processes in multicellular organisms. Here we report two engineered aminoacyl-tRNA synthetases for mammalian bioorthogonal labeling: a tyrosyl ( ScTyr) and a phenylalanyl ( MmPhe) tRNA synthetase that incorporate azide-bearing noncanonical amino acids specifically into the nascent proteomes of host cells. Azide-labeled proteins are chemoselectively tagged via azide-alkyne cycloadditions with fluorophores for imaging or affinity resins for mass spectrometric characterization. Both mutant synthetases label human, hamster, and mouse cell line proteins and selectively activate their azido-bearing amino acids over 10-fold above the canonical. ScTyr and MmPhe label overlapping but distinct proteomes in human cell lines, with broader proteome coverage upon their coexpression. In mice, ScTyr and MmPhe label the melanoma tumor proteome and plasma secretome. This work furnishes new tools for mammalian residue-specific bioorthogonal chemistry, and enables more robust and comprehensive cell-type-specific proteomics in live mammals.
生物正交工具可实现细胞类型特异性蛋白质组学，这是理解多细胞生物中生物学过程的前提。在这里，我们报告了两种用于哺乳动物生物正交标记的工程化氨酰-tRNA 合成酶：一种酪氨酸（ScTyr）和一种苯丙氨酸（MmPhe）tRNA 合成酶，它们可以将带有叠氮化物的非天然氨基酸特异性掺入宿主细胞的新生蛋白质组中。带有叠氮化物的蛋白质通过叠氮化物-炔烃环加成反应与荧光团进行化学选择性标记，用于成像，或与亲和树脂进行质谱特征分析。两种突变合成酶都可标记人、仓鼠和小鼠细胞系的蛋白质，并选择性地激活其叠氮化物修饰的氨基酸，其活性是天然氨基酸的 10 倍以上。ScTyr 和 MmPhe 在人细胞系中标记重叠但不同的蛋白质组，当它们共同表达时，蛋白质组的覆盖范围更广。在小鼠中，ScTyr 和 MmPhe 标记黑色素瘤肿瘤蛋白质组和血浆分泌组。这项工作为哺乳动物残基特异性生物正交化学提供了新的工具，并使活体动物中更稳健和全面的细胞类型特异性蛋白质组学成为可能。
