Purification of Violaxanthin De-epoxidase expressed in E.Coli and identification of disulfide bonds using MS

Sammanfattning: Plants need light to convert earbon dioxide to organic compounds, but when exposed to too much light the photosynthetic machinery takes damage. This is prevented by conversion of violaxanthin to zeaxanthin, which participate in a process that converts the excess light into heat. The conversion of violaxanthin to zeaxanthin is done by the enzyme violaxanthin de-epoxidase. VDE from spinach have been sequenced and expressed in E. coli. After harvesting of periplasmic proteins VDE has been purified using DEAE column, two-step precipitation and lipid affinity precipitation with MGDG. By not reducing VDE while running it on a SDS-PAGE gel and avoiding reducing substances in purification methods and sample preparation for mass spectroscopy the disulfide bonds should be intact. When analyzing non-reduced VDE with MS new mass peaks should appear, representing masses of two peptides linked tagether with a disulfide bond. These peaks were found in the spectrum using Peptidemap. Then these peaks were further analyzed with MS/MS and data interpreted with xQuest. Four disulfide bonds could be confirmed with MS/MS according to xQuest.