Hybrid Triple Quadrupole Mass Spectrometer for Quantitative Mass Spectrometric Ap

描述（由申请人提供）：本申请的目的是获得混合三重四极杆-离子阱质谱仪，特别是来自Applied Biosystems的QTRAP 5500，其具有独特的能力，可支持旨在发现儿科疾病关键生理途径和原因的积极进行中的研究。该仪器将由9名NIH资助的研究人员在Joseph Stokes，Jr.专门用于定量质谱应用。费城儿童医院研究所。这些研究人员发起的正在进行的项目采用基因组发现平台，生物化学，分子，不同的质谱和正交实验来发现感兴趣的蛋白质，翻译后修饰，蛋白水解加工和执行生物功能的蛋白质网络。因此，所有这些项目都很好地推进了发现阶段，并且已经确定了特定和实用的蛋白质和肽靶标。这些项目面临的直接挑战是超越定性描述，通过量化蛋白质和肽的变化来深入探索这些发现。这个目标不能用典型的基于抗体的蛋白质印迹、ELISA或甚至用我们目前的线性离子阱质谱仪来实现，因为这些方法由于缺乏灵敏度、特异性和/或通量而通常既不可行也不实用。靶向肽定量作为一种高选择性、高灵敏度的方法学手段，正成为定量评价蛋白质动态变化、蛋白质网络、翻译后修饰、生物标志物验证和利用的明确选择。为了实现这一目标，PI和合作研究者与拟定仪器的用户合作，正在积极开发采用稳定同位素标记肽和多反应监测（MRM）工作流程的方法，用于定量人体样本以及动物和细胞模型系统中的蛋白质。多反应监测正在成为定量液相色谱串联质谱（LC/MS/MS）实验的标准技术;然而，这些MRM工作流程与我们目前的离子阱平台的生成和应用是有限的。因此，所提出的质谱仪的安装和使用对于实现靶向肽定量方法的开发和实施以产生相当大的新知识和见解至关重要。总体而言，拟议的项目，虽然生物多样性是统一的需要，开发，验证和实施定量假设驱动的质谱方法。因此，虽然每个研究项目在科学价值和完整性方面都是独立的，但这一合并提案的附加值是巨大的。 公共卫生相关性：该仪器将对9名NIH资助的研究人员的研究计划产生重大和直接的影响，这些研究人员从事基础和转化研究，旨在揭示费城儿童医院疾病的基本生理过程和机制。所要求的仪器将有助于量化潜在的新型生物标志物，用于儿科疾病的预后和诊断。

DESCRIPTION (provided by applicant): The purpose of this application is to acquire a hybrid triple quadrupole-ion trap mass spectrometer, specifically the QTRAP 5500 from Applied Biosystems, with unique capabilities to support actively ongoing studies that aim to uncover critical physiological pathways and causes of pediatric diseases. This instrument will be used exclusively for quantitative mass spectrometric applications by 9 NIH funded investigators at the Joseph Stokes, Jr. Research Institute of the Children's Hospital of Philadelphia. Ongoing projects initiated by these investigators have employed genomic discovery platforms, biochemical, molecular, different mass spectrometric and orthogonal experiments to discover proteins of interest, posttranslational modifications, proteolytic processing and networks of proteins that execute biological functions. Therefore all these projects are well advanced past the stages of discovery and specific and practical protein and peptide targets have been identified. The immediate challenge for these projects is to move beyond qualitative descriptions to explore these discoveries in depth by quantifying changes in proteins and peptides. This goal cannot be accomplished with typical antibody based western blot, ELISA or even with our current linear ion trap mass spectrometers since these approaches are often neither feasible nor practical due to lack of sensitivity, specificity, and/or throughput. Targeted peptide quantification is becoming the clear choice as a highly selective and sensitive methodological approach for quantitative evaluation of dynamic changes of proteins, protein networks, posttranslational modifications, validation and utilization of biological markers. Towards this goal the PI and co-Investigator in collaboration with the users of the proposed instrument are actively developing methodologies that employ stable isotope labeled peptides and multiple reaction monitoring (MRM) workflows for the quantification of proteins in human samples as well as in animal and cellular model systems. Multiple reaction monitoring is emerging as the standard technique for quantitative liquid chromatography tandem mass spectrometry (LC/MS/MS) experiments; however generation and application of these MRM workflows with our current ion trap platforms is limited. Therefore the installation and use of the proposed mass spectrometer is vital for enabling the development and implementation of methodologies for targeted peptide quantification to generate sizeable new knowledge and insights. Overall, the proposed projects although biologically diverse are united by the need to develop, validate and implement quantitative hypothesis-driven mass spectrometric approaches. Thus, while each research project stands alone in scientific value and integrity the added value of this combined proposal is substantial. PUBLIC HEALTH RELEVANCE: The instrument will have a major and immediate impact on the research programs of 9 NIH-funded investigators that are engaged in both basic and translational research aimed to uncover fundamental physiological processes and mechanisms of disease at the Children's Hospital of Philadelphia. The requested instrumentation will facilitate the quantification of potentially novel biological markers for the prognosis and diagnosis of pediatric diseases.