Multifunctional reagents for proteomic analysis

描述（由申请人提供）：已经认识到，经历体内老化或患有年龄依赖性病理的组织的蛋白质组学表征必须包括氧化损伤引起的翻译后蛋白质修饰的特定鉴定和定位。3-硝基酪氨酸（3-NY）在细胞内蛋白上的积累是氧化应激伴随生物衰老和年龄相关病理的标志。相反，通过其他更具体的方法，如质谱（MS），在生物样品中定位3-NY到特定蛋白质序列往往失败，因为3-NY修饰的蛋白质丰度低和/或从这些蛋白质中回收含有3-NY的肽的能力差。本提案的具体目标是开发简单的化学衍生化策略，该策略允许对复杂蛋白质混合物中含有3-NY的序列进行选择性修饰，目标如下：(a)选择性富集含3- ny肽，(b)灵敏检测富集的含3- ny肽序列，(c)从不同生物来源获得的含3- ny肽的比较定量，以及(d)开发一种质谱友好的标签，允许产生特定离子，可用于灵敏监测和序列信息。这种方法的直接好处是，科学家在进行组织中3-NY的蛋白质组学分析时，不需要受到高度专业化和昂贵的质谱仪器（如FT-ICR质谱）的限制。我们的方法将允许选择性地富集和监测含有3-NY的蛋白质和/或从这些蛋白质衍生的肽，通过更频繁的访问，更专业的质谱仪器，并允许在氧化蛋白修饰的分析更高的实验生产率。我们的目标将在三个特定目标中实现：特定目标1：筛选老化组织（脑，心脏，骨骼肌）中含有3- ny的蛋白质；特定目标2：开发氧化还原敏感、多功能荧光和同位素编码亲和标签，用于生物样品中含3- ny肽的选择性富集和定量分析；具体目标3：应用新的氧化还原敏感和荧光同位素编码亲和标签对老化组织（脑、心脏、骨骼肌）中含有3- ny的蛋白进行定量分析和测序。

DESCRIPTION (provided by applicant): 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).