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G.-F. Jang, A. M. Crabb, L. Zhang, J. W. Crabb; Improved Methods for Quantifying Plasma Protein Advanced Glycation End Products as Biomarkers for Age-Related Macular Degeneration. Invest. Ophthalmol. Vis. Sci. 2010;51(13):4113.
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Carboxymethyllysine (CML) and pentosidine, two advanced glycation end products (AGEs) elevated in plasma proteins from donors with age-related macular degeneration (AMD), offer biomarker potential for predicting AMD susceptibility and for monitoring therapeutic efficacy (2009 Mol Cell Proteomics 8, 1921-1933). To facilitate quantification of these AGEs in plasma, we have developed improved chromatography methods.
Blood was collected from clinically documented AMD and age-matched normal, healthy donors at the Cole Eye Institute, Cleveland Clinic Foundation. Plasma proteins were precipitated with acetone and hydrolyzed in 6N HCl to determine amounts of protein, furosine, CML and pentosidine. Protein was quantified by AccQ·TagTM amino acid analysis. CML, pentosidine and furosine were fractionated using an Acquity Ultra Performance LC system. CML and furosine were quantified by LC MS/MS using multiple reaction monitoring (MRM) and a Sciex API 3000 triple quadrupole mass spectrometer. Pentosidine was measured by UPLC fluorescence monitoring (excitation = 310 nm; emission = 375 nm).
Analysis methods were modified in several ways relative to the published methods cited above. (1) A Waters UPLC system replaced an Agilent 1100 HPLC. (2) The TFA solvent concentration for chromatography using a HypercarbTM column was increased to 0.2% to improve retention of CML. (3) A Waters HSS C18 column (1.8 µ particles) and aqueous acetonitrile/heptafluorobutyric acid solvents replaced the previously employed aqueous normal phase column in the second chromatography. This decreased analysis time and improved peak shape and resolution of CML, pentosidine, pyridylethylcysteine (internal standard) and furosine. (4) Furosine, a marker of early glycation, was quantified by LC MS/MS MRM.
The technical modifications we have incorporated in our AGEs analysis system improve resolution and quantitative accuracy and decrease analysis time. Application of the improved technology in our AMD biomarker studies will be presented.
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