Shared UPLC-MS/MS for Absolute Quantitative Proteomics

描述(由申请人提供)：为北卡罗来纳大学教堂山分校药学院的一组NIH支持的研究人员申请一种共享仪器--NanUPLC-ESI-MS/MS。使用该仪器的经验丰富的小组将主要使用它对目标蛋白质进行绝对定量分析，特别是那些与药物和代谢物处置有关的蛋白质，如代谢酶和运输蛋白质。其他使用者将使用有针对性的绝对定量蛋白质组学方法，在动物模型中跟踪通过脂复合物传递的人类蛋白质，并评估在病毒识别中调节肝素硫化的多种磺基转移酶的表达。靶向绝对定量蛋白质组学方法远优于目前使用的半定量蛋白质组学方法，因为它具有动态范围大、灵敏度等于或更高、准确度和精密度更好、耗时更少的特点。这些方法现在有时用于专门的蛋白质组实验室，现在可以更广泛地应用于通常熟悉小分子定量LC-MS/MS的实验室，具有高通量，其中经常使用稳定的同位素标准。因此，当生物医学科学家常规使用该方法时，该方法将产生更大的影响，这些科学家可以使用共享的设施。定向绝对定量蛋白质组学将允许我们的NIH支持的研究实验室以前无法使用的实验方法，从而通过能够测量和量化几乎任何蛋白质的组织或生物体中的蛋白质表达，而不是测量间接或不太相关的测量，来推进他们的科学。此外，使用稳定同位素标记的多肽作为标准，将使这些方法更容易在实验室之间进行比较。定向绝对定量蛋白质组学是一种令人兴奋的方法，它将很快成为主流，这里要求的共享仪器将推动北卡罗来纳大学教堂山的这一努力，并建立可以在大学和一般科学界广泛传播和使用的方法。 相关性(对于普通公众)目前检测主要蛋白质的方法耗时长，而且往往不可靠，这些蛋白质可以清除体内的药物，或者将药物输送到体内的目标部位。所要求的仪器将以更省时和更可重复性的方式检测和量化其中的几种蛋白质。使用该仪器的研究人员将探索与药物代谢和基因传递相关的蛋白质的表达和传递，从而提高我们对药物-药物相互作用的理解和我们治疗疾病的能力。

DESCRIPTION (provided by applicant): A shared instrument, nanoUPLC-ESI-MS/MS, is requested for a focused group of NIH supported researchers in the School of Pharmacy at the University of North Carolina at Chapel Hill. The experienced group using the instrument will employ it primarily for absolute quantitative analysis of targeted proteins, especially those proteins involved in drug and metabolite disposition, such as enzymes of metabolism and transport proteins. Other Users will employ targeted absolute quantitative proteomic methods to follow human proteins delivered via lipoplexes in animal models, and to assess the expression of multiple sulfotransferases that modulate heparan sulfation in viral recognition. The targeted absolute quantitative proteomics approach is far superior to presently employed semi-quantitative Western blotting, since it has a large dynamic range, equal or greater sensitivity, better accuracy and precision, and is less time consuming. These methods, now sometimes employed in dedicated proteomics labs, can now be employed more widely, with high throughput, in labs commonly familiar with small molecule quantitative LC-MS/MS where stable isotope standards are routinely employed. Thus, the method will have a greater impact when it is used routinely by biomedical scientists with access to a shared facility. Targeted absolute quantitative proteomics will allow experimental approaches previously unavailable to our NIH supported research labs, thus advancing their science by being able to measure and quantify protein expression in tissue, or the organism directly, for almost any protein rather than measuring indirect or poorly correlated measures such as mRNA. Moreover, using stable isotope labeled peptides as standards will permit the methods to be compared between labs more easily. Targeted absolute quantitative proteomics is an exciting method that will quickly become mainstream, and the shared instrument requested herein will drive this effort at UNC Chapel Hill and establish methods that can be broadly disseminated and employed within the University and the general scientific community. Relevance (for the lay public) Current methods to detect major proteins that eliminate drugs from the body, or deliver drugs to targeted sites in the body, are time-consuming and often unreliable. The requested instrument will detect and quantify several of these proteins in a much more time-efficient and reproducible manner. The researchers using the instrument will explore the expression and delivery of proteins associated with drug metabolism and gene delivery, thus improving our understanding of drug-drug interactions and our ability to treat diseases.