Proteomic analysis of protein nitration in rat cerebellum: effect of biological aging

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Abstract
3‐Nitrotyrosine (3‐NT) is a useful biomarker of increasing oxidative stress and protein nitration during biological aging. The proteomic analysis of cerebellar homogenate from Fisher 344/Brown Norway (BN/F1) rats shows an age‐dependent increase in protein nitration, monitored by western‐blot analysis after two‐dimensional gel electrophoresis (2DE), mainly in the acidic region. Analysis of in‐gel digests by nanoelectrospray (NSI)‐MS/MS resulted in the identification of 16 putatively nitrated proteins. The selective isolation of nitrated proteins using immunoprecipitation, followed by SDS‐PAGE and in‐gel digest/NSI‐MS/MS analysis led to the identification of 22 putatively nitrated proteins, of which 7 were identical to those detected after 2DE. When proteins were separated by solution isoelectrofocusing and analyzed by NSI MS/MS, we obtained MS/MS spectra of 3‐NT containing peptides of four proteins – similar to ryanodine receptor 3, low density lipoprotein related receptor 2, similar to nebulin‐related anchoring protein isoform C and 2,3 cyclic nucleotide 3‐phosphodiesterase. Although the functional consequences of protein nitration for these targets are not yet known, our proteomic experiments serve as a first screen for the more targeted analysis of nitrated proteins from aging cerebellum for functional characterization.

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