Department of Chemistry and Biochemistry, Florida Atlantic University, Boca Raton, Florida 33431, USA.
J Am Chem Soc. 2010 Jan 27;132(3):980-8. doi: 10.1021/ja9043196.
Cell penetrating peptides (CPPs) have attracted recent interest as drug delivery tools, although the mechanisms by which CPPs are internalized by cells are not well-defined. Here, we report a new experimental approach for the detection and secondary structure determination of CPPs in live cells using Raman microscopy with heavy isotope labeling of the peptide. As a first demonstration of principle, penetratin, a 16-residue CPP derived from the Antennapedia homeodomain protein of Drosophila, was measured in single, living melanoma cells. Carbon-13 labeling of the Phe residue of penetratin was used to shift the intense aromatic ring-breathing vibrational mode from 1003 to 967 cm(-1), thereby enabling the peptide to be traced in cells. Difference spectroscopy and principal components analysis (PCA) were used independently to resolve the Raman spectrum of the peptide from the background cellular Raman signals. On the basis of the position of the amide I vibrational band in the Raman spectra, the secondary structure of the peptide was found to be mainly random coil and beta-strand in the cytoplasm, and possibly assembling as beta-sheets in the nucleus. The rapid entry and almost uniform cellular distribution of the peptide, as well as the lack of correlation between peptide and lipid Raman signatures, indicated that the mechanism of internalization under the conditions of study was probably nonendocytotic. This experimental approach can be used to study a wide variety of CPPs as well as other classes of peptides in living cells.
细胞穿透肽 (CPPs) 作为药物传递工具引起了最近的关注，尽管 CPP 被细胞内化的机制尚未明确。在这里，我们报告了一种使用重同位素标记肽的拉曼显微镜检测和确定活细胞中 CPP 二级结构的新实验方法。作为原理的第一个演示，我们在单个活黑素瘤细胞中测量了源自果蝇触角足域蛋白的 16 个残基 CPP penetratin。通过对 penetratin 中的 Phe 残基进行碳-13 标记，将强芳环呼吸振动模式从 1003 移至 967 cm(-1)，从而使肽能够在细胞中被追踪。差谱和主成分分析 (PCA) 分别用于从细胞的背景拉曼信号中解析肽的拉曼光谱。根据酰胺 I 振动带在拉曼光谱中的位置，发现肽的二级结构在细胞质中主要为无规卷曲和β-折叠，在核中可能组装为β-片层。肽的快速进入和几乎均匀的细胞分布，以及肽和脂质拉曼特征之间缺乏相关性，表明在研究条件下内化的机制可能是非内吞作用。这种实验方法可用于研究各种 CPP 以及其他类别的肽在活细胞中的作用。
