DOI QR코드

DOI QR Code

Effect of centrifugation on tryptic protein digestion

  • Kim, Soohwan (Department of Chemistry, Chungnam National University) ;
  • Kim, Yeoseon (Department of Chemistry, Chungnam National University) ;
  • Lee, Dabin (Department of Chemistry, Chungnam National University) ;
  • Kim, Inyoung (Department of Chemistry, Chungnam National University) ;
  • Paek, Jihyun (Department of Chemistry, Chungnam National University) ;
  • Shin, Dongwon (Department of Chemistry, Chungnam National University) ;
  • Kim, Jeongkwon (Department of Chemistry, Chungnam National University)
  • Received : 2017.01.29
  • Accepted : 2017.02.17
  • Published : 2017.04.25

Abstract

This study investigated the effect of centrifugation on tryptic digestion. This was done by applying different centrifugation speeds (6,000, 8,000, 10,000, 20,000, and $30,000{\times}g$) over various durations (0, 10, 20, 30, 40, 50, and 60 min) to digest two model proteins - cytochrome c and myoglobin. The intact proteins and resulting peptides were identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Centrifugation greatly improved the tryptic digestion efficiency of cytochrome c, where either an increase in centrifugation speed or in digestion duration significantly improved the digestion of cytochrome c. However, centrifugation did not noticeably improve the digestion of myoglobin; 16 h of centrifuge-assisted tryptic digestion at $30,000{\times}g$ barely removed the myoglobin protein peak. Similar results were also obtained when using conventional tryptic digestion with gentle mixing. When acetonitrile (ACN) was added to make 10% ACN buffer solutions, the myoglobin protein peak disappeared after 6 h of digestion using both centrifuge-assisted and conventional tryptic digestions.

Keywords

centrifuge;centrifugation;trypsin;digestion;proteins;MALDI

Acknowledgement

Supported by : Chungnam National University

References

  1. W. Y. Chen and Y. C. Chen, Anal. Chem., 79(6), 2394- 2401 (2007). https://doi.org/10.1021/ac0614893
  2. W. Sun, S. Gao, L. Wang, Y. Chen, S. Wu, X. Wang, D. Zheng, and Y. Gao, Mol. Cell. Proteomics, 5(10), S2-S2 (2006). https://doi.org/10.1074/mcp.R500014-MCP200
  3. D. Lopez-Ferrer, J. L. Capelo, and J. Vazquez, J. Proteome Res., 4(5), 1569-1574 (2005). https://doi.org/10.1021/pr050112v
  4. H.-J. Yang, S. Shin, J. Kim, J. Hong, S. Lee, and J. Kim, Rapid Commun. Mass Spectrom., 25(1), 88-92 (2011). https://doi.org/10.1002/rcm.4840
  5. H. J. Yang, J. Hong, S. Lee, S. Shin, and J. Kim, Rapid Commun. Mass Spectrom., 24(7), 901-908 (2010). https://doi.org/10.1002/rcm.4467
  6. D. Lopez-Ferrer, K. Petritis, K. K. Hixson, T. H. Heibeck, R. J. Moore, M. E. Belov, D. G. Camp, and R. D. Smith, J. Proteome Res., 7(8), 3276-3281 (2008). https://doi.org/10.1021/pr7008077
  7. S. Park, T. Kim, J. Lee, M. Seo, and J. Kim, Rapid Commun. Mass Spectrom., 27(7), 842-846 (2013). https://doi.org/10.1002/rcm.6508