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NIH支持的研究人员重点小组申请共享仪器nanoUPLC-ESI-MS/MS。使用该仪器的经验丰富的团队将主要将其用于目标蛋白质的绝对定量分析，特别是那些涉及药物和代谢物处置的蛋白质，如代谢酶和转运蛋白。其他用户将采用靶向绝对定量蛋白质组学方法，在动物模型中跟踪通过脂丛传递的人类蛋白质，并评估在病毒识别中调节肝素硫酸化的多种硫转移酶的表达。靶向绝对定量蛋白质组学方法远优于目前使用的半定量Western blotting，因为它具有大的动态范围，相同或更高的灵敏度，更好的准确性和精密度，并且耗时更少。这些方法现在有时用于专门的蛋白质组学实验室，现在可以更广泛、高通量地应用于通常熟悉小分子定量LC-MS/MS的实验室，这些实验室通常采用稳定同位素标准。因此，当生物医学科学家能够使用共享设施时，这种方法将产生更大的影响。有针对性的绝对定量蛋白质组学将允许以前NIH支持的研究实验室无法使用的实验方法，从而通过能够直接测量和量化组织或生物体中几乎任何蛋白质的蛋白质表达而不是测量间接或不相关的测量（如mRNA）来推进他们的科学。此外，使用稳定同位素标记的肽作为标准将使实验室之间的方法更容易进行比较。靶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，本文所要求的共享仪器将推动北卡罗来纳大学教堂山分校的这项工作，并建立可以在大学和一般科学界广泛传播和使用的方法。

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.