Browse > Article
http://dx.doi.org/10.5012/bkcs.2011.32.2.599

Pressure Cycling Technology-assisted Protein Digestion for Efficient Proteomic Analysis  

Choi, Hyun-Su (Division of Mass Spectrometry, Korea Basic Science Institute)
Lee, Sang-Kwang (Division of Mass Spectrometry, Korea Basic Science Institute)
Kwon, Kyung-Hoon (Division of Mass Spectrometry, Korea Basic Science Institute)
Yoo, Jong-Shin (Division of Mass Spectrometry, Korea Basic Science Institute)
Ji, Kelly (CM Corporation Ltd.)
Kim, Jin-Young (Division of Mass Spectrometry, Korea Basic Science Institute)
Publication Information
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 $50^{\circ}C$. 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 $25^{\circ}C$ 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 $37^{\circ}C$ for 16h, the PCT-assisted digestion with urea at $25^{\circ}C$ for 1 h, and the non-PCT-assisted digestion with urea at $25^{\circ}C$ 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.
Keywords
Fast digestion; Pressure cycling technology (PCT); Enzyme activity; Stem cell proteome analysis;
Citations & Related Records

Times Cited By Web Of Science : 2  (Related Records In Web of Science)
Times Cited By SCOPUS : 2
연도 인용수 순위
  • Reference
1 Kim, S. S.; Choi, J. M.; Kim, J. W.; Ham, D. S.; Ghil, S. H.; Kim, M. K.; Kim-Kwon, Y.; Hong, S. Y.; Ahn, S. C.; Kim, S. U.; Lee, Y. D.; Kim, H. S. Neuroreport 2005, 16, 1357.   DOI   ScienceOn
2 Carvalho, P. C.; Xu, T.; Han, X.; Cociorva, D.; Barbosa, V. C.; Yates, J. R. Mol. Cell. Proteomics 2006, 5, S174.
3 Lippincott, J.; Apostol, L. Anal. Biochem. 1999, 267, 57.   DOI   ScienceOn
4 Tagwerker, C.; Flick, K.; Cui, M.; Guerrero, C.; Dou, Y.; Auer, B.; Baldi, P.; Huang, L.; Kaiser, P. Mol. Cell. Proteomics 2006, 5, 737.
5 Klammer, A. A.; MacCoss, M. J. J. Proteome. Res. 2006, 5, 695.   DOI   ScienceOn
6 Rogalski, J. C.; Lin, M. S.; Sniatynski, M. J.; Taylor, T. J.; Youhnovski, N.; Przybylski, M.; Kast, J. J. Am. Soc. Mass. Spectrom. 2005, 16, 505.   DOI   ScienceOn
7 Washburn, M. P.; Wolters, D.; Yates, J. R. Nat. Biotechnol. 2001, 19, 242.   DOI   ScienceOn
8 Hernandez, P.; Muller, M.; Appel, R. D. Mass. Spectrom. Rev. 2006, 25, 235.   DOI   ScienceOn
9 Lopez-Ferrer, D.; Petritis, K.; Hixson, K. K.; Heibeck, T. H.; Moore, R. J.; Belov, M. E.; Camp II, D. G; Smith, R. D. J. Proteome. Res. 2008, 7, 3276.   DOI   ScienceOn
10 Rabilloud, T.; Adessi, C.; Giraudel, A.; Lunardi, J. Electrophoresis 1997, 18, 307.   DOI   ScienceOn
11 McCarthy, J.; Hopwood, F.; Oxley, D.; Laver, M.; Castagna, A.; Righetti, R. G.; Williams, K.; Herbert, B. J. Proteome. Res. 2003, 2, 239.   DOI   ScienceOn
12 Hervey, W. J. T.; Strader, M. B.; Hurst, G. B. J. Proteome. Res. 2007, 6, 3054.   DOI   ScienceOn
13 Blonder, J.; Chan, K. C.; Issaq, H. J.; Veenstra, T. D. Nat. Protoc. 2006, 1, 2784.
14 Zhong, H.; Zhang, Y.; Wen, Z.; Li, L. Nat. Biotechnol. 2004, 22, 1291.   DOI   ScienceOn
15 Lopez-Ferrer, D.; Capelo, J. L.; Vazquez, J. J. Proteome. Res. 2005, 4, 1569.   DOI   ScienceOn
16 Pramanik, B. N.; Mirza, U. A.; Ing, Y. H.; Liu, Y. H.; Bartner, P. L.; Weber, P. C.; Bose, A. K. Protein Sci. 2002, 11, 2676.
17 Sun, W.; Gao, S.; Wang, L.; Chen, Y.; Wu, S.; Wang, X.; Zheng, D.; Gao, Y. Mol. Cell. Proteomics 2006, 5, 769.
18 Chicon, R.; Belloque, J.; Recio, I.; Lopez-Fandino, R. J. Dairy Res. 2006, 73, 121.   DOI   ScienceOn
19 Ringham, H.; Bell, R. L.; Smejkal, G. B.; Behnke, J.; Witzmann, F. A. Electrophoresis 2007, 28, 1022.   DOI   ScienceOn
20 Meersman, F.; Dobson, C. M.; Heremans, K. Chem. Soc. Rev. 2006, 35, 908.   DOI   ScienceOn
21 Belloque, J.; Chicon, R.; Lopez-Fandino, R. J. Agric. Food Chem. 2007, 55, 5282.   DOI   ScienceOn
22 Yang, H.-J.; Hong, J.; Lee, S.; Shin, S.; Kim, J.; Kim, J. Rapid Commun. Mass Spectrom. 2010, 24, 901.   DOI   ScienceOn
23 Hu, Q.; Noll, R. J.; Li, H.; Makarov, A.; Hardman, M.; Graham, C. R. J. Mass. Spectrom. 2005, 40, 430.   DOI   ScienceOn
24 Blackler, A. R.; Klammer, A. A.; MacCoss, M. J.; Wu, C. C. Anal. Chem. 2006, 78, 1337.   DOI   ScienceOn
25 Denison, C.; Rudner, A. D.; Gerber, S. A.; Bakalarski, C. E.; Moazed, D.; Gygi, S. P. Mol. Cell. Proteomics 2005, 4, 246.   DOI   ScienceOn