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Bulletin of the Korean Chemical Society (BKCS)

ISSN 0253-2964(Print)
ISSN 1229-5949(Online)
Volume 32, Number 2
BKCSDE 32(2)
February 20, 2011 

 
Title
Pressure Cycling Technology-assisted Protein Digestion for Efficient Proteomic Analysis
Author
Hyunsu Choi, Sang Kwang Lee, Kyung Hoon Kwon, Jong Shin Yoo, Kelly Ji, Jin Young Kim*
Keywords
Fast digestion, Pressure cycling technology (PCT), Enzyme activity, Stem cell proteome 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 oC. 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 oC 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 oC for 16h, the PCT-assisted digestion with urea at 25 oC for 1 h, and the non-PCT-assisted digestion with urea at 25 oC 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.
Page
599 - 604
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