Proteomic analysis of protein nitration in rat cerebellum: effect of biological aging.
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.
Gokulrangan
Giridharan
G
Department of Pharmaceutical Chemistry, University of Lawrence, Kansas 66047, USA.
Zaidi
Asma
A
Michaelis
Mary L
ML
Schöneich
Christian
C
D000375:Aging / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002531:Cerebellum / Q000378:metabolism*; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D011506:Proteins / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*; D051381:Rats; D011916:Rats, Inbred F344; D014443:Tyrosine / Q000031:analogs & derivatives / Q000378:metabolism
D011506:Proteins; C002744:3-nitrotyrosine; D014443:Tyrosine