Digestion completeness of microwave-assisted and conventional trypsin-catalyzed reactions.
Microwave-assisted proteolytic digestion often yields misscleaved peptides, attributed to incomplete hydrolysis reactions between enzymes and substrates. The number of missed cleavages is an important parameter in proteome database searching. This study investigates how various factors affect digestion processes. Optimum conditions for microwave-assisted digestion (50 mM Tris buffer, 30 min at 60 degrees C, and enzyme to protein molar ratio of 1:5) were determined. The digestion products obtained from eight standard proteins were characterized based on matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Experimental results indicate that the digestion temperature, reaction time, enzyme to substrate ratio, and digestion buffer affect the number of misscleaved peptides and incomplete digestion percentages. Although all protein molecules in a sample could be digested into peptides within a few minutes under microwave irradiation, longer reaction times or methods to maximize the enzyme activity should be considered if digestion completeness is a major concern.
Reddy
P Muralidhar
PM
Department of Chemistry, National Dong Hwa University, Hualien, Taiwan.
Hsu
Wan-Yu
WY
Hu
Jun-Fu
JF
Ho
Yen-Peng
YP
D002384:Catalysis / Q000528:radiation effects; D008872:Microwaves; D011829:Radiation Dosage; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D014357:Trypsin / Q000737:chemistry* / Q000528:radiation effects*
D014357:Trypsin