Research report Proteomic identification of proteins oxidized by Ah(1–42) in synaptosomes: Implications for Alzheimer’s disease Debra Boyd-Kimball a , Alessandra Castegna a , Rukhsana Sultana a , H. Fai Poon a , Robin Petroze a , Bert C. Lynn a,b , Jon B. Klein d , D. Allan Butterfield a,c, * a Department of Chemistry, Center for Membrane Sciences, University of Kentucky, Lexington, KY 40506-0055, USA b Core Proteomics Laboratory, University of Kentucky, Lexington, KY 40506-0055, USA c Sanders-Brown Center on Aging, University of Kentucky, Lexington, KY 40506-0055, USA d Kidney Disease Program and Proteomics Core Laboratory, University of Louisville School of Medicine and VAMC, Louisville, KY 40292, USA Accepted 24 February 2005 Available online 15 April 2005 Abstract Protein oxidation has been implicated in Alzheimer’s disease (AD) and can lead to loss of protein function, abnormal protein turnover, interference with cell cycle, imbalance of cellular redox potential, and eventually cell death. Recent proteomics work in our laboratory has identified specifically oxidized proteins in AD brain such as: creatine kinase BB, glutamine synthase, ubiquitin carboxy-terminal hydrolase L-1, dihydropyrimidase-related protein 2, a-enolase, and heat shock cognate 71, indicating that a number of cellular mechanisms are affected including energy metabolism, excitotoxicity and/or synaptic plasticity, protein turnover, and neuronal communication. Synapse loss is known to be an early pathological event in AD, and incubation of synaptosomes with amyloid beta peptide 1 – 42 (Ah 1 – 42) leads to the formation of protein carbonyls. In order to test the involvement of Ah(1 – 42) in the oxidation of proteins in AD brain, we utilized two-dimensional gel electrophoresis, immunochemical detection of protein carbonyls, and mass spectrometry to identify proteins from synaptosomes isolated from Mongolian gerbils. Ah(1 – 42) treatment leads to oxidatively modified proteins, consistent with the notion that Ah(1 – 42)-induced oxidative stress plays an important role in neurodegeneration in AD brain. In this study, we identified h-actin, glial fibrillary acidic protein, and dihydropyrimidinase-related protein-2 as significantly oxidized in synaptosomes treated with Ah(1 –42). Additionally, H + -transporting two-sector ATPase, syntaxin binding protein 1, glutamate dehydrogenase, g-actin, and elongation factor Tu were identified as increasingly carbonylated. These results are discussed with respect to their potential involvement in the pathogenesis of AD. D 2005 Elsevier B.V. All rights reserved. Theme: Disorders of the nervous system Topic: Degenerative disease: Alzheimer’s-beta amyloid Keywords: Alzheimer’s disease; Amyloid h-peptide (1 – 42); Amyloid h-peptide (42 – 1); Oxidative stress; Proteomics 1. Introduction Alzheimer’s disease (AD) is characterized pathologically by the presence of senile plaques, neurofibrillary tangles, and synapse loss. Additionally, AD is associated with oxidative stress. Amyloid beta peptide is a 39–43 amino acid peptide produced from proteolytic processing of amyloid precursor protein (APP), a ubiquitous transmem- brane glycoprotein [58].Ah(1–42) is the primary compo- nent of the core of senile plaques and has been shown to induce protein oxidation in vitro and in vivo [6– 8,14,28,34,41,49,69].Ah(1 – 42)-induced oxidative stress has been proposed to play a central role in the pathogenesis of AD [8,10,14 – 16]. Consistent with this notion, oxidative 0006-8993/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2005.02.086 * Corresponding author. Department of Chemistry, Center for Mem- brane Sciences, and Sanders-Brown Center on Aging, 121 Chemistry- Physics Building, University of Kentucky, Lexington, KY 40506-0055, USA. Fax: +1 859 257 5876. E-mail address: dabcns@uky.edu (D.A. Butterfield). Brain Research 1044 (2005) 206 – 215 www.elsevier.com/locate/brainres