Proteomic approaches to oxidative protein modifications implicated in the mechanism of aging.
Accumulation of oxidatively modified proteins is widely observed in aged animal tissues. Protein carbonyls are mostly derived from lysine, arginine, proline and threonine residues under oxidative conditions. Many groups have investigated carbonylated proteins since a convenient immunochemical procedure was established for detecting dinitrophenyl derivatives of carbonyls and applied to proteomic research. An alternative method of tagging with biotin or fluorescent dyes has been also introduced to proteomic analysis of protein carbonyls. Nitrotyrosine was primarily identified as a biomarker of cellular damage and inflammation under nitrosative stress. Nitrated proteins have been subsequently detected in aged animal tissues and Alzheimer's disease affected brains by Western blotting, and identified by mass spectrometry. Protein s-thiolation, a mixed-derivatization of cysteine (Cys) by conjugation of low-molecular-weight thiol compounds, is recognized as protecting functional proteins from more serious damage. A method of biotin labeling has been used in proteomics for tracing protein s-thiolation. Among all kinds of amino acid residues, methionine (Met) is the most susceptible to reactive oxygen species, and Met oxidation seems to occur in ordinary cellular circumstances because most cells contain Met sulfoxide reductases, which might prevent serious cellular damage. In proteomic analysis, Met sulfoxide-containing peptides are generally observed as 16-Da-high mass peaks in peptide mass fingerprinting. A modified procedure of two-dimensional gel electrophoresis, in which proteins are kept under non-oxidative conditions throughout the procedure, is appropriate for the estimation of the Met sulfoxide level of each protein in aged animal tissues and cells to evaluate the pathophysiological significance of Met oxidation in the mechanism of aging.
Toda
Tosifusa
T
Research Team for Mechanism of Aging, Tokyo Metropolitan Institute of Gerontology, Tokyo, Japan. ttoda@tmig.or.jp
Nakamura
Megumi
M
Morisawa
Hiraku
H
Hirota
Mikako
M
Nishigaki
Ryuichi
R
Yoshimi
Yoji
Y
D000375:Aging / Q000378:metabolism / Q000502:physiology*; D000544:Alzheimer Disease / Q000378:metabolism; D000818:Animals; D003545:Cysteine / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D006801:Humans; D007525:Isoelectric Focusing; D008715:Methionine / Q000378:metabolism; D018384:Oxidative Stress / Q000502:physiology*; D014176:Protein Biosynthesis / Q000502:physiology; D050050:Protein Carbonylation / Q000502:physiology; D040901:Proteomics; D017382:Reactive Oxygen Species / Q000378:metabolism
D017382:Reactive Oxygen Species; D008715:Methionine; D003545:Cysteine