After elution from the hydroxyapatite column, the OPH fractions were combined and subjected to gel filtration on a Superose12 column using a Biologic Duo Flow protein purification system. Fractions selleck chemical were eluted with 50 mM sodium phosphate buffer, pH 7 containing 1 mM EDTA and 0. 2 M NaCl at a rate of 0. 5 ml min in 0. 5 ml fractions. Fractions that contained OPH activity were combined and stored at 20 C. The pooled semi purified OPH was analyzed by mass spectroscopy to verify that no other esterases or proteases were present. Overexpression of OPH in COS 7 cells COS 7 cells were transfected using TransIT LT1 transfec tion reagent and the vector pCDNA3. 1 encoding OPH with a Flag tag using the transfection reagents manufac turers instructions. COS 7 cells overexpressing OPH were selected using 1 mg ml G418 over a three week period.
Cells surviving selection were termed COS 7 OPH for further experiments and were maintained with 1 mg ml G418. LC MS MS mass spectroscopy Protein bands were individually excised from the n PAGE gel and cut into small pieces using a scalpel. The gel pieces were destained, disulfide bonds reduced, unmodified thiol groups alkylated, and the proteins digested with trypsin overnight using the In Gel Tryptic Digestion Kit according to the manufacturers instructions. After digestion, the liquid containing the peptides from each band was transferred to a 1. 5 ml tube. The peptides were further extracted from each gel piece by covering gel piece with extraction buffer consisting of formic acid acetonitrile water for 10 min then collecting the liquid and adding it to the appropri ate 1.
5 ml tube. The peptides in the vial inserts were completely dried using a DNA Speed Vac Concentrator. Peptides were rehydrated with 0. 1% formic acid and further purified using ZipTipU C18 tips according to manufacturers in structions. Peptides eluted from zip tips were transferred to vial inserts. The peptides in the vial inserts were com pletely dried using the Speed Vac Concentrator and then rehydrated in a volume of 4 ul of formic acid acetonitrile water. A volume of 2 ul of each sample was trapped by a picofrit column packed with C18 and equilibrated in 0. 1% formic acid in water acetonitrile. Peptides were then eluted with a gradient of 2 to 40% of solvent B con taining 0. 1% formic acid in acetonitrile over 60 min at a flow rate of 200 nL min.
Eluted peptides were analyzed by electrospray ionization using a LTQ XL ion trap mass spectrometer. Mass spectrometry data were acquired using data dependent acquisition with a series selleck inhibitor of one full scan followed by a zoom scan and then a MS MS scan of the ions. The dynamic exclusion duration was 30 ms. Proteins were identified from each MS raw data file using the SEQUEST search algorithm and the SwissProt UNIPROT database through the Bioworks browser, version 3. 3.