School of Analytical Sciences Adlershof, Humboldt-Universität zu Berlin, Albert-Einstein-Straße 5-9, 12489 Berlin, Germany.
Department of Chemistry, Humboldt-Universität zu Berlin, Brook-Taylor-Straße 2, 12489 Berlin, Germany.
Anal Chem. 2020 Jun 16;92(12):8553-8560. doi: 10.1021/acs.analchem.0c01404. Epub 2020 Jun 5.
Surface-enhanced Raman scattering (SERS) can provide information on the structure, composition, and interaction of molecules in the proximity of gold nanoparticles, thereby enabling studies of adsorbed biomolecules . Here, the processing of the protein corona and the corresponding protein-nanoparticle interactions in live J774 cells incubated with gold nanoparticles was characterized by SERS. Samples of isolated cytoplasm, devoid of active processing, of the same cell line were used as references. The occurrence of the most important SERS signals was compared in both types of samples. The comparison of signal abundances, supported by multivariate assessment, suggests a decreased nanoparticle-peptide backbone interaction and an increased contribution of denatured proteins in endolysosomal compartments, indicating an interaction of protein fragments with the gold nanoparticles in the endolysosome of the living cells. To study the protein fragmentation in a model and to confirm the assignment of specific spectral signatures in the live cell spectra, SERS data were collected from a solution of bovine serum albumin (BSA) digested by trypsin as an enzymatic model and from solutions of intact BSA and trypsin. The spectra from the enzymatic model confirm the strong interaction of protein fragments with the gold nanoparticles in the endolysosomal compartments. By proteomic analysis, using combined sodium dodecyl sulfate-polyacrylamide gel electrophoresis and high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry of the extracted hard corona, we directly identified protein fragments, some originating from the culture medium. The results illustrate the use of appropriate models for the validation of SERS spectra and have potential implications for further developments of SERS as an analytical and biomedical tool.
表面增强拉曼散射（SERS）可以提供金纳米粒子附近分子的结构、组成和相互作用的信息，从而能够研究吸附的生物分子。在这里，通过 SERS 对金纳米粒子孵育的活 J774 细胞中蛋白质冠的处理和相应的蛋白质-纳米粒子相互作用进行了表征。同系细胞的分离细胞质样本，没有活性处理，被用作参考。比较了两种类型样本中最重要的 SERS 信号的出现。基于多元评估的信号丰度比较表明，纳米颗粒-肽主链相互作用降低，内溶酶体隔间中变性蛋白质的贡献增加，表明蛋白质片段与活细胞内溶酶体中的金纳米粒子相互作用。为了在模型中研究蛋白质片段，并确认活细胞光谱中特定光谱特征的分配，从牛血清白蛋白（BSA）的溶液中收集 SERS 数据，该溶液由胰蛋白酶消化作为酶模型，以及完整的 BSA 和胰蛋白酶溶液。来自酶模型的光谱证实了蛋白质片段与内溶酶体隔间中的金纳米粒子的强烈相互作用。通过蛋白质组学分析，使用联合十二烷基硫酸钠-聚丙烯酰胺凝胶电泳和高效液相色谱-电喷雾电离-串联质谱法对提取的硬壳进行分析，我们直接鉴定了蛋白质片段，其中一些来自培养基。结果说明了使用适当模型验证 SERS 光谱的重要性，并对进一步开发 SERS 作为分析和生物医学工具具有潜在意义。
