S. Poggioli, J. Mary, H. Bakala, B. Friguet

Oxidatively modified proteins have been analyzed in aging human peripheral blood lymphocytes since protein modification by oxidation and other related pathways are believed to contribute to the intracellular age-related accumulation of damaged proteins, a process that has been associated with the cellular functional deficits that occur with age. Advanced glycation end products (AGE) were characterized using an anti-AGE antibody raised against glycated RNAse and a small but significant (40%) increase of protein glycoxidation in cytosolic extracts from old donors (86 to 91 years old) versus young donors (20 to 25 years old) was observed by enzyme-linked immunosorbent assay. To further analyze the pattern of glycoxidized proteins, two dimensional gel electrophoresis followed by western blotting with the same anti-AGE antibody were performed. The protein silver stain and the immunoblot patterns were not superimposable indicating that glycoxidative modifications are targeting only a restricted set of proteins. Among these preferential protein targets, seven of them exhibited a significant age-related increased immunoreactivity with the anti-AGE antibody suggesting that the corresponding modified proteins might serve as biomarkers of aging lymphocytes. Identification of several target proteins has been achieved by mass spectrometry (LC/MS). Other age-related protein modifications such as conjugation with the lipid peroxidation product 4-hydroxy-2-nonenal have also been studied. The patterns of modified proteins has been analyzed using two dimensional gel electrophoresis followed by western blotting with an antibody recognizing 4-hydroxy-2-nonenal protein adducts using the same proteomic approach as for glycoxidative modifications. Specific protein targets for this modification that might serve as biomarkers of aging lymphocytes are currently characterized and identified by mass spectrometry.

Keywords (Optional): 
protein modification
mass spectrometry