Abstract
Purpose: :
To investigate modifications from glycolaldehyde and A2E to the membrane like protein fragment, laminin, as a model for aging in Bruch’s membrane in age related eye diseases using high pressure liquid chromatography-mass spectroscopy (LC-MS) with PDA detection.
Methods: :
Laminin was reacted with either glycolaldehyde or A2E, during irradiation, and then tryptically digested 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).
Results: :
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.
Conclusions: :
This model system has identified a number of specific molecular modifications resulting from the reaction of either A2E or advanced glycation endproducts (AGEs) with components of the BM. These in vitro experiments are essential to identify similar modifications in vivo.
Keywords: Bruch's membrane • aging • oxidation/oxidative or free radical damage