Application of a temperature-controllable microreactor to simple and rapid protein identification using MALDI-TOF MS.
We have carried out a simultaneous thermal denaturation and trypsin digestion of proteins using a temperature-controllable microreactor. This is a simple and rapid sample preparation technique for use before matrix-assisted laser desorption ionization time-of-flight mass spectrometry. In contrast to a conventional sample preparation method, which involves several chemical treatments, our sample preparation was performed using only trypsin digestion with the thermal denaturation of the target protein. Optimization of the reactor operational parameters for trypsin digestion using a temperature-controllable microreactor was carried out. The entire trypsin digestion procedure took about 11 min, and consisted of 1 min for the thermal denaturation of the sample protein (3 microl, 0.2 microM) at 85 degrees C, and 10 min for digestion of the protein at 37 degrees C. The resulting sequence coverage ranged from 24% to 57%, which was sufficient for practical protein identification.
Sim
Tae Seok
TS
School of Electrical Engineering and Computer Science, Seoul National University, 301-1116, San 56-1, Sillim-Dong, Gwanak-Gu, Seoul 151-742, Korea. sim93@snu.ac.kr
Kim
Eun-Mi
EM
Joo
Hwang Soo
HS
Kim
Byung Gee
BG
Kim
Yong-Kweon
YK
D008832:Microchemistry; D046210:Microfluidic Analytical Techniques; D011506:Proteins / Q000032:analysis*; D040901:Proteomics; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D011506:Proteins