A NanoLC-Orbitrap Tribrid Instrument for Comprehensive Proteomics Analyses

项目摘要： 该提案要求为Thermo Orbitrap Fusion Lumos和Ultimate 3000 RSLCnano提供资金 UHPLC仪器可满足高端质谱和蛋白质组学日益增长的需求 在华盛顿大学的仪器。对于成功的人来说，这个工具将是无价的 完成现有NIH资助的项目，需要对蛋白质进行敏感和深入的表征， 翻译后修饰。值得注意的是，我们的几个项目试图确定量化的变化， 磷酸化在特定的大脑区域，需要开发和实施超敏感的新的 这将有助于更广泛的研究界的分析工作流程。成功的关键 我们国家卫生研究院研究项目的一个重要成果是研制出一个集成系统，它能够：1）无人值守 样品注入，真正的纳升流速和压力公差为10，000 psi，2）低于1 ppm的质量精度 在色谱时间尺度上，3）在单个质谱中> 5000的动态范围， 定量和质量测定，4）快速高质量分辨率扫描~ 450，000以分辨同量异位素质量 标签，如NeuCode SILAC中的标签，而5）获得> 15 Hz的高质量串联质谱（MS 2）扫描 与具有四极线性离子阱灵敏度的单独质量分析器并联，6）同步 前体和碎片离子的分离和碎裂，用于改进的特异性和定量准确性， 各种量化方法。华盛顿大学的质谱仪具有这些功能， 目前严重超额认购，并在很大程度上无法获得我们的赠款的用户组。我们建议 共享蛋白质组学仪器的收购将使并推动我们资助的 research.所要求的仪器将使我们的教师，以解决目前的技术限制， 他们各自的领域，改善样品分析瓶颈在华盛顿大学，并大大 扩大NIH未来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.