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Geeng-Fu Jang, Lei Zhang, Li Hong, Hua Wang, Robert G. Salomon, John W. Crabb; Quantification Of CEP By LC MS/MS. Invest. Ophthalmol. Vis. Sci. 2012;53(14):6478.
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© ARVO (1962-2015); The Authors (2016-present)
Carboxyethylpyrrole (CEP) adducts are oxidative modifications derived from docosahexaenoate-containing lipids and elevated in ocular tissues and plasma in age-related macular degeneration (AMD). CEP stimulates angiogenesis in vivo, and mice immunized with CEP develop an AMD-like phenotype. CEP adducts offer AMD biomarker potential, however current ELISA-based CEP assays can yield variable results. To improve accuracy and precision, we are developing an LC MS/MS-based method for quantifying CEP adducts in plasma.
Authentic standards of CEP-lysine and CEP-BSA were prepared by organic synthesis. Blood was collected from AMD and control donors at the Cole Eye Institute and plasma was prepared. Plasma proteins were reduced and alkylated. Because CEP is destroyed by acid hydrolysis, methods were developed for the complete enzymatic digestion of plasma proteins in the presence of butylhydroxytoluene (BHT). Hydrolysis efficiency was evaluated with several combinations of proteases, including pepsin, pronase E, proteinase K, leucine aminopeptidase and prolidase. Protein was quantified by amino acid analysis (AAA). Post cleavage sample clean up and recovery was evaluated with several solid phase extraction (SPE) cartridges. CEP-lysine was quantified by LC MS/MS using a Waters UPLC, a Sciex API 3000 triple quadrupole mass spectrometer and multiple reaction monitoring.
Optimum enzymatic hydrolysis of plasma proteins in the presence of BHT was achieved with pronase E and leucine aminopeptidase over ~24 h. About 90% efficiency of hydrolysis was obtained, as calculated by AAA of the enzymatic hydrolysate before and after HCl hydrolysis. To date, the 1g Hypercarb SPE cartridge has yielded the best recovery of protein and CEP-lysine (~60%) from sample clean up; the 2g Hypercarb cartridges will be tested for improved yield. Excellent resolution of CEP-lysine has been obtained on a Waters HSS C18 column using aqueous acetonitrile/heptafluorobutyric acid solvents. Current results suggest the limits of detection for CEP-lysine by our LC MS/MS system is less than 1 pmol.
We anticipate the technology described above will significantly improve the accuracy and precision of quantifying CEP adducts. Application of the improved technology in our AMD biomarker studies will be presented.
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