Pressure Cycling Technology-assisted Protein Digestion for Efficient Proteomic Analysis

Abstract

In typical proteomic analysis, trypsin digestion is one of the most time-consuming steps. Conventional proteomic sample preparation methods use an overnight trypsin digestion method. In this study, we compared high-pressure cycling technology (PCT) during enzyme digestion for proteome analysis to the conventional method. We examined the effect of PCT on enzyme activity at temperatures of 25, 37, and 50 o C. Although a fast digestion (1 h) was used for the standard protein mixture analysis, the PCT-assisted method with urea showed better results for protein sequence coverage and the number of peptides identified compared with the conventional method. There was no significant difference between temperatures for PCT-assisted digestion; however, we selected PCT-assisted digestion with urea at 25 o C as an optimized method for fast enzyme digestion, based on peptide carbamylation at these conditions. The optimized method was used for stem cell proteome analysis. We identified 233, 264 and 137 proteins using the conventional method with urea at 37 o C for 16h, the PCT-assisted digestion with urea at 25 o C for 1 h, and the non-PCT-assisted digestion with urea at 25 o C for 1 h, respectively. A comparison of these results suggests that PCT enhanced the enzyme digestion by permitting better access to cleavage sites on the proteins.

Recent advances on multidimensional liquid chromatography–mass spectrometry for proteomics: From qualitative to quantitative analysis—A review

“…Although much effort has been made to increase the efficiency of traditional in-solution proteolysis by high pressure [66,67], microwave irradiation [68], infrared radiation [69,70] and laser radiation [71], the online hyphenation of in-solution digestion with HPLC is rather difficult. To solve these problems, much attention has been paid to the development of immobilized enzymatic reactors (IMERs) [72][73][74][75][76].…”