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L. S. Murdaugh, J. P. Dillon, E. R. Gaillard; Non-Enzymatic Nitration of Laminin: A Model for Inflammation at the Level of Bruch’s Membrane. Invest. Ophthalmol. Vis. Sci. 2010;51(13):5230.
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To investigate modifications to a laminin fragment induced by non-enzymatic nitration as a model for inflammation at the level of Bruch’s membrane.
Laminin samples were incubated at 37oC (pH=7.4) in the dark for seven days with either 200 mM NaNO2 or 200 mM NaCl. The samples were dialyzed against 10 mM phosphate buffer until the Greiss assay was negative for nitrite or the equivalent amount of time for the control sample. The samples were then subjected to tryptic digestion before being analyzed by LC-MS (Thermo Finnigan, LCQ Advantage, Surveyor; Surveyor LC with fluorescence and PDA detectors, quadrupole ion trap mass analyzer, electrospray ion source).
Modifications to laminin via the Maillard reaction occurred preferentially to lysine and arginine residues in both the glycolaldehyde and A2E modified laminin samples. The most abundant fragments from the A2E modified samples were consistent with additions of A2E derived aldehydes resulting from cleavages closest to the pyridinium ring in A2E (m/z 592) and oxidized A2E (m/z 608). However, the high reactivity of A2E and A2E derived aldehydes generated numerous modified peptides of lower abundance, which were identified using corresponding MS/MS data and UV-Vis absorbance. These modified peptides were consistent with additions of A2E derived aldehydes, resulting from cleavages along the polyene chain of A2E and oxidized A2E, to different positions within the laminin fragment.
This model system has identified a number of specific molecular modifications resulting from the reaction of either A2E or advanced glycation end-products (AGEs) with components of the BM. These in vitro experiments are essential to identify similar modifications in vivo.
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