Sodium-deoxycholate-assisted tryptic digestion and identification of proteolytically resistant proteins.
Identification of proteolytically resistant proteins with compact molecular structure and/or poor water solubility is a challenge in current proteomic study. In this study, sodium deoxycholate (SDC)-assisted tryptic digestion and identification of proteolytically resistant myoglobin and integral membrane proteins were systematically investigated. When the effect of SDC up to 10% on trypsin activity was investigated, little decrease in the trypsin activity was observed in 1% SDC solution, 2-5% SDC decreased the enzyme activity only by about 13.6%, and even in the presence of 10% SDC trypsin still retained 77.4% of its activity. Matrix-assisted laser desorption ionization time of flight mass spectrometry analysis showed that SDC could be removed from sample solution with acid treatment followed by centrifugation, and the remaining SDC, if any, had little effect on mass spectrometry analysis with regard to the number and signal/noise ratio of ions in the mass spectra. Compared with urea and methanol, two other commonly used additives in addition to SDS in proteomic analysis, SDC improved more efficiently the denaturation, solubilization, and tryptic digestion of proteins, particularly proteolytically resistant myoglobin and integral membrane proteins, thereby enhancing the efficiency of their identification with regard to the number of identified proteins and unique peptides and the sequence coverage of matched proteins.
Lin
Yong
Y
College of Life Sciences, Hunan Normal University, Changsha, Hunan 410081, PR China.
Zhou
Jian
J
Bi
Deng
D
Chen
Ping
P
Wang
Xianchun
X
Liang
Songping
S
D000818:Animals; D001120:Arginine / Q000031:analogs & derivatives / Q000378:metabolism; D045424:Complex Mixtures / Q000737:chemistry; D003840:Deoxycholic Acid / Q000737:chemistry / Q000494:pharmacology*; D006868:Hydrolysis / Q000187:drug effects; D057927:Hydrophobic and Hydrophilic Interactions; D007700:Kinetics; D013058:Mass Spectrometry; D008565:Membrane Proteins / Q000378:metabolism*; D000432:Methanol / Q000494:pharmacology; D009211:Myoglobin / Q000378:metabolism*; D010455:Peptides / Q000378:metabolism; D011489:Protein Denaturation / Q000187:drug effects; D012995:Solubility; D014357:Trypsin / Q000378:metabolism*; D014508:Urea / Q000494:pharmacology
D045424:Complex Mixtures; D008565:Membrane Proteins; D009211:Myoglobin; D010455:Peptides; D003840:Deoxycholic Acid; D014508:Urea; D001120:Arginine; C009727:benzoylarginine ethyl ester; D014357:Trypsin; D000432:Methanol