Women and Children's Hospital Affiliated to Xiamen University, School of medicine, College of Chemistry and Chemical Engineering, College of Energy, Xiamen University, Xiamen 361005, China.
Anal Chem. 2022 Jan 18;94(2):1318-1324. doi: 10.1021/acs.analchem.1c04531. Epub 2021 Dec 20.
Human pepsin is a digestive protease that plays an important role in the human digestive system. The secondary structure of human pepsin determines its bioactivity. Therefore, an in-depth understanding of human pepsin secondary structure changes is particularly important for the further improvement of the efficiency of human pepsin biological function. However, the complexity and diversity of the human pepsin secondary structure make its analysis difficult. Herein, a convenient method has been developed to quickly detect the secondary structure of human pepsin using a portable Raman spectrometer. According to the change of surface-enhanced Raman spectroscopy (SERS) signal intensity and activity of human pepsin at different pH values, we analyze the change of the human pepsin secondary structure. The results show that the content of the β-sheet gradually increased with the increase in the pH in the active range, which is in good agreement with circular dichroism (CD) measurements. The change of the secondary structure improves the sensitivity of human pepsin SERS detection. Meanwhile, human pepsin is a commonly used disease marker for the noninvasive diagnosis of gastroesophageal reflux disease (GERD); the detection limit of human pepsin we obtained is 2 μg/mL by the abovementioned method. The real clinical detection scenario is also simulated by spiking pepsin solution in saliva, and the standard recovery rate is 80.7-92.3%. These results show the great prospect of our method in studying the protein secondary structure and furthermore promote the application of SERS in clinical diagnosis.
人胃蛋白酶是一种消化蛋白酶，在人体消化系统中发挥着重要作用。人胃蛋白酶的二级结构决定了它的生物活性。因此，深入了解人胃蛋白酶二级结构的变化对于进一步提高人胃蛋白酶生物功能的效率尤为重要。然而，人胃蛋白酶二级结构的复杂性和多样性使其分析变得困难。在此，我们开发了一种使用便携式拉曼光谱仪快速检测人胃蛋白酶二级结构的简便方法。根据人胃蛋白酶在不同 pH 值下表面增强拉曼散射（SERS）信号强度和活性的变化，我们分析了人胃蛋白酶二级结构的变化。结果表明，在活性范围内，随着 pH 的增加，β-折叠的含量逐渐增加，这与人胃蛋白酶圆二色性（CD）测量结果一致。二级结构的变化提高了人胃蛋白酶 SERS 检测的灵敏度。同时，人胃蛋白酶是胃食管反流病（GERD）非侵入性诊断中常用的疾病标志物；我们通过上述方法获得的人胃蛋白酶检测限为 2 μg/mL。通过在唾液中添加胃蛋白酶溶液模拟真实的临床检测场景，并得到了 80.7-92.3%的标准回收率。这些结果表明，我们的方法在研究蛋白质二级结构方面具有广阔的前景，并进一步推动了 SERS 在临床诊断中的应用。
