Rothenberg Daniel A, Taliaferro J Matthew, Huber Sabrina M, Begley Thomas J, Dedon Peter C, White Forest M
Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; The Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Biochemistry and Molecular Genetics, University of Colorado School of Medicine, Aurora, CO 80045, USA; RNA Bioscience Initiative, University of Colorado School of Medicine, Aurora, CO 80045, USA.
iScience. 2018 Nov 30;9:367-381. doi: 10.1016/j.isci.2018.11.004. Epub 2018 Nov 5.
To quantify dynamic protein synthesis rates, we developed MITNCAT, a method combining multiplexed isobaric mass tagging with pulsed SILAC (pSILAC) and bio-orthogonal non-canonical amino acid tagging (BONCAT) to label newly synthesized proteins with azidohomoalanine (Aha), thus enabling high temporal resolution across multiple conditions in a single analysis. MITNCAT quantification of protein synthesis rates following induction of the unfolded protein response revealed global down-regulation of protein synthesis, with stronger down-regulation of glycolytic and protein synthesis machinery proteins, but up-regulation of several key chaperones. Waves of temporally distinct protein synthesis were observed in response to epidermal growth factor, with altered synthesis detectable in the first 15 min. Comparison of protein synthesis with mRNA sequencing and ribosome footprinting distinguished protein synthesis driven by increased transcription versus increased translational efficiency. Temporal delays between ribosome occupancy and protein synthesis were observed and found to correlate with altered codon usage in significantly delayed proteins.
为了量化动态蛋白质合成速率，我们开发了MITNCAT，这是一种将多重等压质量标记与脉冲稳定同位素标记氨基酸（pSILAC）以及生物正交非天然氨基酸标记（BONCAT）相结合的方法，用于用叠氮高丙氨酸（Aha）标记新合成的蛋白质，从而能够在单次分析中跨多种条件实现高时间分辨率。对未折叠蛋白反应诱导后的蛋白质合成速率进行MITNCAT定量分析，结果显示蛋白质合成整体下调，糖酵解和蛋白质合成机制蛋白的下调更为明显，但几种关键伴侣蛋白上调。在对表皮生长因子的反应中观察到了时间上不同的蛋白质合成波，在前15分钟内就能检测到合成的变化。将蛋白质合成与mRNA测序和核糖体足迹分析进行比较，区分了由转录增加与翻译效率提高驱动的蛋白质合成。观察到核糖体占据与蛋白质合成之间的时间延迟，并发现其与显著延迟的蛋白质中密码子使用的改变相关。
