Rapid and Simple Analysis of the Human Pepsin Secondary Structure Using a Portable Raman Spectrometer.

Abstract

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.