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.
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 beta-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 neurodegenerative disorders without the limitations of the immunochemical detection method.
Castegna
Alessandra
A
Department of Chemistry, Center of Membrane Sciences, University of Kentucky, Lexington 40506-0055, USA.
Aksenov
Michael
M
Aksenova
Marina
M
Thongboonkerd
Visith
V
Klein
Jon B
JB
Pierce
William M
WM
Booze
Rosemarie
R
Markesbery
William R
WR
Butterfield
D Allan
DA
D000544:Alzheimer Disease / Q000378:metabolism*; D016564:Amyloid beta-Protein Precursor / Q000378:metabolism; D000911:Antibodies, Monoclonal / Q000276:immunology; D015153:Blotting, Western; D001923:Brain Chemistry; D003402:Creatine Kinase / Q000737:chemistry* / Q000502:physiology; D052276:Creatine Kinase, BB Form; D015180:Electrophoresis, Gel, Two-Dimensional; D005609:Free Radicals; D005974:Glutamate-Ammonia Ligase / Q000737:chemistry* / Q000276:immunology / Q000502:physiology; D018698:Glutamic Acid / Q000378:metabolism; D005973:Glutamine / Q000378:metabolism; D006801:Humans; D007091:Image Processing, Computer-Assisted; D007527:Isoenzymes / Q000737:chemistry* / Q000502:physiology; D009410:Nerve Degeneration; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000502:physiology; D010084:Oxidation-Reduction; D040901:Proteomics; D012394:Rosaniline Dyes; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013194:Staining and Labeling; D013869:Thiolester Hydrolases / Q000737:chemistry* / Q000502:physiology; D043222:Ubiquitin Thiolesterase
D016564:Amyloid beta-Protein Precursor; D000911:Antibodies, Monoclonal; D005609:Free Radicals; D007527:Isoenzymes; D009419:Nerve Tissue Proteins; D012394:Rosaniline Dyes; D005973:Glutamine; D018698:Glutamic Acid; C048139:Coomassie blue; D003402:Creatine Kinase; D052276:Creatine Kinase, BB Form; D013869:Thiolester Hydrolases; D043222:Ubiquitin Thiolesterase; D005974:Glutamate-Ammonia Ligase