Original Contribution PROTEOMIC IDENTIFICATION OF OXIDATIVELY MODIFIED PROTEINS IN ALZHEIMER’S DISEASE BRAIN. PART I: CREATINE KINASE BB, GLUTAMINE SYNTHASE, AND UBIQUITIN CARBOXY-TERMINAL HYDROLASE L-1 ALESSANDRA CASTEGNA,* MICHAEL AKSENOV, † MARINA AKSENOVA, ‡ VISITH THONGBOONKERD, § JON B. KLEIN, § WILLIAM M. PIERCE,  ROSEMARIE BOOZE, † WILLIAM R. MARKESBERY, ¶ and D. ALLAN BUTTERFIELD* ¶ *Department of Chemistry, Center of Membrane Sciences, † Departments of Anatomy and Neurobiology and ‡ Pharmacology, University of Kentucky, Lexington, KY, USA; § Kidney Disease Program and Core Proteomics Laboratory and  Department of Pharmacology, University of Louisville School of Medicine and VAMC, Louisville, KY, USA; and ¶ Sanders-Brown Center on Aging, University of Kentucky, Lexington, KY, USA (Received 5 February 2002; Revised 30 April 2002; Accepted 9 May 2002) Abstract—Oxidative alterations of proteins by reactive oxygen species (ROS) have been implicated in the progression of aging and age-related neurodegenerative disorders such as Alzheimer’s disease (AD). Protein carbonyls, a marker of protein oxidation, are increased in AD brain, indicating that oxidative modification of proteins is relevant in AD. Oxidative damage can lead to several events such as loss in specific protein function, abnormal protein clearance, depletion of the cellular redox-balance and interference with the cell cycle, and, ultimately, to neuronal death. Identification of specific targets of protein oxidation represents a crucial step in establishing a relationship between oxidative modification and neuronal death in AD, and was partially achieved previously in our laboratory through immunochemical detection of creatine kinase BB and -actin as specifically oxidized proteins in AD brain versus control brain. However, this process is laborious, requires the availability of specific antibodies, and, most importantly, requires a reasonable guess as to the identity of the protein in the first place. In this study, we present the first proteomics approach to identify specifically oxidized proteins in AD, by coupling 2D fingerprinting with immunological detection of carbonyls and identification of proteins by mass spectrometry. The powerful techniques, emerging from application of proteomics to neurodegenerative disease, reveal the presence of specific targets of protein oxidation in Alzheimer’s disease (AD) brain: creatine kinase BB, glutamine synthase, and ubiquitin carboxy-terminal hydrolase L-1. These results are discussed with reference to potential involvement of these oxidatively modified proteins in neurodegeneration in AD brain. Proteomics offers a rapid means of identifying oxidatively modified proteins in aging and age-related neurode- generative disorders without the limitations of the immunochemical detection method. © 2002 Elsevier Science Inc. Keywords—Free radicals, Proteomics, Protein oxidation, Alzheimer’s disease, Mass spectrometry, Oxidative stress INTRODUCTION Oxidative alterations of proteins by reactive oxygen spe- cies (ROS) or other reactive substances have been im- plicated in the progression of aging and age-related neu- rodegenerative disorders, among which the most prevalent is Alzheimer’s disease (AD) [1–5]. Protein carbonyls, whose formation is considered a detectable marker of protein oxidation, are increased in AD [6], and their level is higher where the histopathology of the disease is more pronounced [7]. Protein carbonyl forma- tion might be due to ROS-mediated oxidation of amino acid side chains or by covalent binding to products of lipid peroxidation or glycoxidation [4,8]. The resulting chemical modifications appear to be involved in cellular metabolism. Oxidative damage can lead to loss in spe- cific protein function [9]. Once oxidized, proteins can be more prone to degradation by specific proteases [9] un- less the oxidative modification induces protein aggrega- tion, which conversely results in deposition of proteoly- sis-resistant protein aggregates [10,11]. In addition, protein oxidation products could affect cell growth and Address correspondence to: Professor D. Allan Butterfield, Depart- ment of Chemistry and Center of Membrane Sciences, University of Kentucky, Lexington, KY 40506-0055, USA; Tel: (859) 257-3184; Fax: (859) 257-5876; E-Mail: dabcns@uky.edu. Free Radical Biology & Medicine, Vol. 33, No. 4, pp. 562–571, 2002 Copyright © 2002 Elsevier Science Inc. Printed in the USA. All rights reserved 0891-5849/02/$–see front matter PII S0891-5849(02)00914-0 562