Enhanced proteomic analysis of Streptomyces peucetius cytosolic protein using optimized protein solubilization protocol.
Improvements in the dissolution of proteins in two-dimensional gel electrophoresis have greatly advanced the ability to analyze the proteomes of microorganisms under a wide variety of physiological conditions. This study examined the effect of various combinations of chaotropic agents, a reducing agent, and a detergent on the dissolution of the Streptomyces peucetius cytosolic proteins. The use of urea alone in a rehydration buffer as a chaotropic agent gave the proteome a higher solubility than any of the urea and thiourea combinations, and produced the highest resolution and clearest background in two-dimensional gel electrophoresis. Two % CHAPS, as a detergent in a rehydration buffer, improved the protein solubility. After examining the effect of several concentrations of reducing agent, 50 mM DTT in a rehydration buffer was found to be an optimal condition for the proteome analysis of Streptomyces. Using this optimized buffer condition, more than 2,000 distinct and differentially expressed soluble proteins could be resolved using two-dimensional gel electrophoresis with a pI ranging from 4-7. Under this optimized condition, 15 novel small proteins with low-level expression, which could not be analyzed under the non-optimized conditions, were identified. Overall, the optimized condition helped produce a better reference gel for Streptomyces peucetius.
Lee
Kwang Won
KW
Interdisciplinary Program for Biochemical Engineering and Biotechnology, Institute of Molecular Biology and Genetics, Seoul National University, Seoul, Korea.
Song
Eunjung
E
Kim
June-Hyung
JH
Lee
Hei Chan
HC
Liou
Kwang Kyoung
KK
Sohng
Jae Kyung
JK
Kim
Byung-Gee
BG
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification*; D003600:Cytosol / Q000737:chemistry; D003902:Detergents; D004229:Dithiothreitol; D015180:Electrophoresis, Gel, Two-Dimensional; D040901:Proteomics / Q000379:methods; D012995:Solubility; D021241:Spectrometry, Mass, Electrospray Ionization; D013302:Streptomyces / Q000737:chemistry* / Q000235:genetics; D053719:Tandem Mass Spectrometry
D001426:Bacterial Proteins; D003902:Detergents; D004229:Dithiothreitol