Biological Nanochemistry Research Group, Institute of Materials and Environmental Chemistry, Research Centre for Natural Sciences, Magyar tudósok körútja 2, Budapest, 1117, Hungary.
Plasma Chemistry Research Group, Institute of Materials and Environmental Chemistry, Research Centre for Natural Sciences, Magyar tudósok körútja 2, Budapest, 1117, Hungary.
Anal Bioanal Chem. 2020 Jul;412(19):4619-4628. doi: 10.1007/s00216-020-02711-8. Epub 2020 May 29.
Extracellular vesicles (EVs) are lipid bilayer-bounded particles that are actively synthesized and released by cells. The main components of EVs are lipids, proteins, and nucleic acids and their composition is characteristic to their type and origin, and it reveals the physiological and pathological conditions of the parent cells. The concentration and protein composition of EVs closely relate to their functions; therefore, total protein determination can assist in EV-based diagnostics and disease prognosis. Here, we present a simple, reagent-free method based on attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy to quantify the protein content of EV samples without any further sample preparation. After calibration with bovine serum albumin, the protein concentration of red blood cell-derived EVs (REVs) were investigated by ATR-FTIR spectroscopy. The integrated area of the amide I band was calculated from the IR spectra of REVs, which was proportional to the protein quantity in the sample' regardless of its secondary structure. A spike test and a dilution test were performed to determine the ability to use ATR-FTIR spectroscopy for protein quantification in EV samples, which resulted in linearity with R values as high as 0.992 over the concentration range of 0.08 to 1 mg/mL. Additionally, multivariate calibration with the partial least squares (PLS) regression method was carried out on the bovine serum albumin and EV spectra. R values were 0.94 for the calibration and 0.91 for the validation set. The results indicate that ATR-FTIR measurements provide a reliable method for reagent-free protein quantification of EVs. Graphical abstract.
细胞外囊泡 (EVs) 是由细胞主动合成并释放的具有双层脂膜的颗粒。EVs 的主要成分是脂质、蛋白质和核酸，其组成特征与其类型和来源有关，反映了母细胞的生理和病理状态。EVs 的浓度和蛋白质组成与其功能密切相关；因此，总蛋白测定可辅助基于 EV 的诊断和疾病预后。在这里，我们提出了一种简单的、无试剂的方法，基于衰减全反射傅里叶变换红外（ATR-FTIR）光谱，无需进一步的样品制备即可定量 EV 样品中的蛋白质含量。用牛血清白蛋白进行校准后，用 ATR-FTIR 光谱研究了红细胞衍生的 EV（REV）的蛋白质浓度。从 REV 的红外光谱中计算酰胺 I 带的积分面积，其与样品中的蛋白质数量成正比，而与蛋白质的二级结构无关。进行了加标测试和稀释测试，以确定 ATR-FTIR 光谱在 EV 样品中的蛋白质定量能力，结果表明，在 0.08 至 1 mg/mL 的浓度范围内，线性度高达 0.992，R 值很高。此外，还对牛血清白蛋白和 EV 光谱进行了偏最小二乘（PLS）回归的多元校准。校准和验证集的 R 值分别为 0.94 和 0.91。结果表明，ATR-FTIR 测量为 EV 无试剂蛋白质定量提供了一种可靠的方法。
