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由氧化损伤引起的翻译后蛋白质修饰的特异性鉴定和定位。3-硝基酪氨酸（3-NY）在细胞内蛋白质上的积累是伴随生物衰老和年龄相关病理学的氧化应激的标志。相反，通过其他更特异的方法如质谱法（MS）将生物样品中的3-NY定位到特异性蛋白质序列常常失败，这是由于3-NY修饰的蛋白质的丰度低和/或从这些蛋白质中回收含3-NY的肽的能力差。该提案的具体目标是开发简单的化学衍生化策略，其允许选择性修饰复杂蛋白质混合物中含有3-NY的序列，具有以下目标：（a）选择性富集含3-NY的肽，（B）灵敏检测富集的含3-NY的肽序列，（c）从不同生物来源获得的含3-NY的肽的比较定量，和（d）开发质谱友好的标签以允许产生特定离子，其可用于灵敏的监测和序列信息。这种方法的直接好处是，科学家将不需要受到高度专业化和昂贵的MS仪器（如FT-ICR MS）的限制，以进行组织中3-NY的蛋白质组学分析。我们的方法将允许选择性富集和监测3-NY含有蛋白质和/或肽衍生自这些蛋白质更频繁地访问，不太专业的MS仪器，并允许更高的实验生产率在氧化蛋白质修饰的分析。我们的目标将在三个具体目标中实现：具体目标1：在衰老组织中筛选含3-NY的蛋白质（脑、心脏、骨骼肌）;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富集和定量分析含3-NY的肽;具体目标3：新的氧化还原敏感的和荧光同位素编码的亲和标记物在老化组织（脑、心脏、骨骼肌）中含3-NY的蛋白质的定量分析和测序中的应用。

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).