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KMID : 0385520170300020096
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Analytical Science & Technology
2017 Volume.30 No. 2 p.96 ~ p.101
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Effect of centrifugation on tryptic protein digestion
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Kim Soo-Hwan
Kim Yeo-Seon
Lee Da-Bin
Kim In-Young
Paek Ji-Hyun
Shin Dong-Won
Kim Jeong-Kwon
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Abstract
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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×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×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 centrifugeassisted
and conventional tryptic digestions.
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KEYWORD
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centrifuge, centrifugation, trypsin, digestion, proteins, MALDI
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