Multifunctional reagents for proteomic analysis

描述(申请人提供)：已经认识到，正在经历体内衰老或遭受年龄相关病理的组织的蛋白质组学特征必须包括由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)在细胞内蛋白质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，由于3-NY修饰的蛋白质丰度低和/或含有3-NY的多肽从这些蛋白质中回收得很差，通过其他更具体的方法，如质谱仪(MS)，在生物样品中定位3-NY到特定蛋白质序列的方法往往失败。这一建议的具体目标是开发简单的化学衍生化策略，以实现对复杂蛋白质混合物中3-NY序列的选择性修饰，目的如下：(A)选择性地富集3-NY-多肽，(B)灵敏地检测富含3-NY的多肽序列，(C)对从不同生物来源获得的3-NY-多肽进行比较定量，以及(D)开发一种质谱学友好的标记，以产生可用于敏感监测和序列信息的特定离子。这种方法的直接好处是，科学家不需要受到限制使用高度专业化和昂贵的MS仪器(如FT-ICR MS)来进行组织中3-NY的蛋白质组学分析。我们的方法将允许通过更频繁、更不专业的MS仪器选择性地浓缩和监测含有3-NY的蛋白质和/或从这些蛋白质衍生的肽，并允许在分析氧化蛋白质修饰时获得更高的实验生产率。我们的目标将通过三个具体目标实现：具体目标1：筛选衰老组织(脑、心脏、骨骼肌)中含3-NY的蛋白质；具体目标2：开发氧化还原敏感的多功能荧光和同位素编码的亲和标签，用于从生物样品中选择性地浓缩和定量分析含3-NY的多肽；具体目标3：应用新的氧化还原敏感和荧光同位素编码的亲和标签对衰老组织(脑、心脏、骨骼肌)中含3-NY的蛋白质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).