Department of Chemistry, Columbia University, New York, NY 10027, USA.
Proc Natl Acad Sci U S A. 2013 Jul 9;110(28):11226-31. doi: 10.1073/pnas.1303768110. Epub 2013 Jun 24.
Synthesis of new proteins, a key step in the central dogma of molecular biology, has been a major biological process by which cells respond rapidly to environmental cues in both physiological and pathological conditions. However, the selective visualization of a newly synthesized proteome in living systems with subcellular resolution has proven to be rather challenging, despite the extensive efforts along the lines of fluorescence staining, autoradiography, and mass spectrometry. Herein, we report an imaging technique to visualize nascent proteins by harnessing the emerging stimulated Raman scattering (SRS) microscopy coupled with metabolic incorporation of deuterium-labeled amino acids. As a first demonstration, we imaged newly synthesized proteins in live mammalian cells with high spatial-temporal resolution without fixation or staining. Subcellular compartments with fast protein turnover in HeLa and HEK293T cells, and newly grown neurites in differentiating neuron-like N2A cells, are clearly identified via this imaging technique. Technically, incorporation of deuterium-labeled amino acids is minimally perturbative to live cells, whereas SRS imaging of exogenous carbon-deuterium bonds (C-D) in the cell-silent Raman region is highly sensitive, specific, and compatible with living systems. Moreover, coupled with label-free SRS imaging of the total proteome, our method can readily generate spatial maps of the quantitative ratio between new and total proteomes. Thus, this technique of nonlinear vibrational imaging of stable isotope incorporation will be a valuable tool to advance our understanding of the complex spatial and temporal dynamics of newly synthesized proteome in vivo.
新蛋白质的合成是分子生物学中心法则的关键步骤，是细胞在生理和病理条件下快速响应环境信号的主要生物学过程。然而，尽管沿着荧光染色、放射自显影和质谱等方向进行了广泛的努力，但在活系统中以亚细胞分辨率选择性地可视化新合成的蛋白质组仍然具有挑战性。在此，我们报告了一种通过利用新兴的受激拉曼散射（SRS）显微镜结合氘标记氨基酸的代谢掺入来可视化新生蛋白质的成像技术。作为首次演示，我们以高时空分辨率在活哺乳动物细胞中对新合成的蛋白质进行了成像，而无需固定或染色。通过这种成像技术，可以清楚地识别出具有快速蛋白质周转的亚细胞区室，如 HeLa 和 HEK293T 细胞中的亚细胞区室，以及分化神经元样 N2A 细胞中的新生长的神经突。从技术上讲，氘标记氨基酸的掺入对活细胞的干扰最小，而细胞沉默拉曼区中外源碳-氘键（C-D）的 SRS 成像具有高度的灵敏度、特异性，并且与活细胞系统兼容。此外，与总蛋白质组的无标记 SRS 成像相结合，我们的方法可以轻松生成新蛋白质组与总蛋白质组之间定量比率的空间图谱。因此，这种稳定同位素掺入的非线性振动成像技术将是深入了解体内新合成蛋白质组复杂时空动力学的有价值的工具。
