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Proteomic analysis of protein nitration in rat cerebellum: effect of biological aging
Author(s) -
Gokulrangan Giridharan,
Zaidi Asma,
Michaelis Mary L.,
Schöneich Christian
Publication year - 2007
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1111/j.1471-4159.2006.04334.x
Subject(s) - chemistry , proteomics , biochemistry , immunoprecipitation , microbiology and biotechnology , gel electrophoresis , western blot , nitration , nitrotyrosine , gene isoform , cerebellum , biology , gene , enzyme , nitric oxide synthase , organic chemistry , neuroscience
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.