A NanoLC-Orbitrap Tribrid Instrument for Comprehensive Proteomics Analyses

项目总结： 该提案要求为Thermo Orbitrap Fusion Lumos和旗舰3000 RSLCNano提供资金 UHPLC仪器满足日益增长的高端质谱学和蛋白质组学需求 华盛顿大学的仪器。这一工具对成功的 完成美国国立卫生研究院资助的现有项目，要求对蛋白质和 翻译后修饰。值得注意的是，我们的几个项目试图确定 特定脑区的磷酸化，需要开发和实施超敏感的新技术 对更广泛的研究界有用的分析工作流程。对成功的关键 我们NIH研究项目的完成是一个能够无人值守的集成系统的可用性 样品注入，真正的纳升流速和10000磅/平方英寸的压力容差，2)质量精度低于1ppm 在层析时间刻度上，3)在单个质谱图中的动态范围为&gt；5000，以实现准确 定量和质量测定，4)快速高质量分辨率扫描~450,000以解析等压质量 与NeuCode SILAC中的标签类似，5)获取高质量串联质谱图(MS2)扫描15赫兹 与独立的质量分析器并行，具有四极线性离子陷阱的灵敏度，同步 前体和碎片离子分离和碎裂可提高特异性和定量准确性 各种量化方法。华盛顿大学具有这些能力的质谱仪是 目前订阅量严重超额，基本上无法提供给我们授权的用户群。我们建议， 收购共享蛋白质组学仪器将使我们能够并推动成功完成我们资助的 研究。所要求的仪器将允许我们的教员解决当前技术方面的限制 他们各自的领域，改善了华盛顿大学的样本分析瓶颈，并大大 扩大未来NIH资助项目的范围和能力。

Project Summary: This proposal requests funds for a Thermo Orbitrap Fusion Lumos and an Ultimate 3000 RSLCnano UHPLC instrument to meet increasing demand for high-end mass spectrometry and proteomics instrumentation at the University of Washington. This instrument will be invaluable for the successful completion of existing NIH funded projects requiring sensitive and in-depth characterization of proteins and post-translational modifications. Notably, several of our projects seek to identify quantitative changes in phosphorylation in specific brain regions, requiring the development and implementation of ultra-sensitive new analytical workflows that would be useful for the broader research community. Critical to the successful completion of our NIH research projects is the availability of an integrated system capable of 1) unattended sample injection, true nanoliter flow rates and pressure tolerance to 10,000 psi, 2) sub 1 ppm mass accuracy on a chromatographic time-scale, 3) a dynamic range of > 5000 in a single mass spectrum for accurate quantification and mass determination, 4) rapid high mass resolution scans ~ 450,000 to resolve isobaric mass labels like those in NeuCode SILAC, while 5) acquiring high quality tandem mass spectra (MS2) scans > 15 Hz in parallel with a separate mass analyzer with sensitivity of a quadrupole linear ion trap, 6) synchronous precursor and fragment ion isolation and fragmentation for improved specificity and quantitative accuracy in various quantification methods. Mass spectrometers with these capabilities at the University of Washington are currently heavily oversubscribed and largely unavailable to our grant’s group of users. We propose that the acquisition of the shared proteomics instrument will enable and drive the successful completion of our funded research. The requested instrumentation will allow our faculty to address current technological limitations in their respective fields, improve the sample analysis bottlenecks at the University of Washington, and greatly expand the scope and capabilities of future NIH funded projects.