The reactive aldehyde, 4-hydroxynonenal (HNE), is a product of lipid peroxidation that can covalently modify and inactivate proteins. Previously, we reported increased HNE modification of select retinal proteins resolved by one-dimensional gel electrophoresis in aged Fisher 344 × Brown Norway rats (Louie, J.L., Kapphahn, R.J., Ferrington, D.A., 2002. Proteasome function and protein oxidation in the aged retina. Exp. Eye Res. 75, 271-284). In the current study, quantitative assessment of HNE molar content using slot blot immunoassays showed HNE content is increased 30% in aged rat retina. In contrast, there was no age-related difference in HNE content in individual spots resolved by 2D gel electrophoresis suggesting the increased modification is likely on membrane proteins that are missing on 2D gels. The HNE-immunoreactive proteins resolved by 2D gel electrophoresis were identified by MALDI-TOF mass spectrometry. These proteins are involved in metabolism, chaperone function, and fatty acid transport. Proteins that were frequently modified and had the highest molar content of HNE included triosephosphate isomerase, α enolase, heat shock cognate 70 and βB2 crystallin. Immunochemical detection of HNE adducts on retinal sections showed greater immune reaction in ganglion cells, photoreceptor inner segment, and the inner plexiform layer. Identification of HNE modified proteins in two alternative model systems, human retinal pigment epithelial cells in culture (ARPE19) and human donor eyes, indicated that triosephosphate isomerase and α enolase are generally modified. These results identify a common subset of proteins that contain HNE adducts and suggest that select retinal proteins are molecular targets for HNE modification.
Bibliographical noteFunding Information:
The authors would like to thank the personnel at the Minnesota Lions Eye Bank for their assistance in acquiring eyes and the Mass Spectrometry Consortium for the Life Sciences, University of Minnesota, St. Paul, MN, for assistance in acquisition and interpretation of mass spectral data. We acknowledge the Core Grant for Visual Research (National Institutes of Health grant EY11374), which funded the facility for retinal histology. We are grateful to Linda McLoon, PhD for assistance in rat perfusion and acquisition of histology images and Curt Nordgaard for manuscript editing. We would also like to thank Tina Tran and Elizabeth Peters for their technical assistance, and David Bernlohr, PhD for the FABP5 antibody. This work was supported by: National Institutes of Health grants (AG19024, EY013623 and EY014176 (DAF), AG025392 (TWO)), Foundation Fighting Blindness, American Federation for Aging Research, Minnesota Medical Foundation, Grant-In-Aid from the Graduate School of the University of Minnesota, and an unrestricted grant to the Department of Ophthalmology from Research to Prevent Blindness Foundation. K.M.B was supported by Undergraduate Research Opportunities at the University of Minnesota.
- mass spectrometry
- two-dimensional gel electrophoresis