It has been realized that a proteomic characterization of tissue undergoing in vivo aging or suffering age-dependent pathologies must include the specific identification and localization of post-translational protein modifications resulting from oxidative damage. The accumulation of 3-nitrotyrosine (3-NY) on intracellular proteins is a hallmark of oxidative stress accompanying biological aging and age-related pathologies. Instead, the location of 3-NY in biological samples to specific protein sequences by other, more specific methods such as mass spectrometry (MS) have often failed due to the low abundance of 3-NY-modified proteins and/or the poor recovery of 3-NY-containing peptides from these proteins. The specific goals of this proposal are the development of simple chemical derivatization strategies, which allow for the selective modification of 3-NY containing sequences in complex protein mixtures with the following goals: (a) selective enrichment of 3-NY-containing peptides, (b) sensitive detection of the enriched 3-NY-containing peptide sequences, (c) comparative quantification of 3-NY-containing peptides obtained from different biological sources, and (d) development of a mass spectrometry-friendly tag to allow the generation of specific ions, which can be used for sensitive monitoring and sequence information. The immediate benefit of such methodology is that scientists would not need to be limited by restricted access to highly specialized and expensive MS instrumentation (such as FT-ICR MS) to conduct proteomic analysis of 3-NY in tissue. Our methodology would allow selectively enrichment and monitoring of 3-NY containing proteins and/or peptides derived from these proteins by more frequently accessible, less specialized MS instrumentation, and allow for higher experimental productivity in the analysis of oxidative protein modifications. Our objectives will be achieved in three Specific Aims:
Specific Aim 1 : Screening for 3-NY-containing proteins in aging tissue (brain, heart, skeletal muscle);
Specific Aim 2 : Development of redox-sensitive, multifunctional fluorescent and isotope-coded affinity tags for the selective enrichment and quantitative analysis of 3-NY-containing peptides from biological samples;
Specific Aim 3 : Application of the new redox-sensitive and fluorescent isotope-coded affinity tags to the quantitative analysis and sequencing of 3-NY containing proteins in aging tissue (brain, heart, skeletal muscle). ? ?
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