Selection of thiol- and disulfide-containing proteins of Escherichia coli on activated thiol-Sepharose.
Activated thiol-Sepharose (ATS) facilitates selection of thiol-containing proteins. In control- and menadione-treated Escherichia coli, batch selection performed under denaturing conditions revealed distinct two-dimensional electrophoresis (2DE) patterns. Using shotgun proteomics, 183 thiol-containing proteins were identified in control ATS-selected extracts and 126 were identified in menadione-treated E. coli, with 85 proteins being common to both. More than 90% of identified proteins contained one or more cysteines. Blocking with N-ethyl maleimide followed by reduction facilitated ATS-based selection of disulfide-containing proteins. In total, 62 proteins were unique to control cells and 164 were identified in menadione-treated E. coli cells, with 29 proteins being common to both. Proteins from menadione-treated cells were excised from 2DE gels, digested with trypsin, and identified by peptide mass fingerprinting. This revealed 19 unique proteins, 14 of which were identified by shotgun proteomics. Outer membrane proteins A, C, W, and X and 30S ribosomal protein S1 were found in 2DE but not by shotgun proteomics. Foldases, ribosomal proteins, aminoacyl transfer RNA (tRNA) synthetases, and metabolic and antioxidant enzymes were prominent among identified proteins, and many had previously been found to respond to, and be targets for, oxidative stress in E. coli. ATS provides a convenient and rapid way to select thiol-containing proteins.
Hu
Wentao
W
Proteomics Research Group, Department of Biochemistry, University College Cork, Cork, Ireland.
Tedesco
Sara
S
McDonagh
Brian
B
Bárcena
José Antonio
JA
Keane
Catherine
C
Sheehan
David
D
D004220:Disulfides; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000187:drug effects / Q000254:growth & development; D029968:Escherichia coli Proteins / Q000032:analysis / Q000737:chemistry* / Q000302:isolation & purification*; D016877:Oxidants / Q000494:pharmacology; D040901:Proteomics; D012685:Sepharose; D013438:Sulfhydryl Compounds; D024483:Vitamin K 3 / Q000494:pharmacology
D004220:Disulfides; D029968:Escherichia coli Proteins; D016877:Oxidants; D013438:Sulfhydryl Compounds; D024483:Vitamin K 3; D012685:Sepharose