A NanoLC-Orbitrap Tribrid Instrument for Comprehensive Proteomics Analyses

项目概要： 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 华盛顿大学的仪器。该工具对于成功的 完成现有的 NIH 资助项目，需要对蛋白质和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 特定大脑区域的磷酸化，需要开发和实施超灵敏的新方法 对更广泛的研究界有用的分析工作流程。对成功至关重要 我们 NIH 研究项目的完成取决于集成系统的可用性，该系统能够 1) 无人值守 样品注射，真正的纳升流速和压力耐受性高达 10,000 psi，2) 低于 1 ppm 质量精度 在色谱时间尺度上，3) 单质谱图的动态范围 > 5000，可实现准确的分析 定量和质量测定，4) 快速高质量分辨率扫描 ~ 450,000 以解析同量异位素质量 类似 NeuCode SILAC 中的标签，同时 5) 获取高质量串联质谱 (MS2) 扫描 > 15 Hz 与具有四极线性离子阱灵敏度的单独质量分析器并行，6) 同步 前体离子和碎片离子分离和碎裂，以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功能的质谱仪是 目前已经严重超额认购，并且我们的赠款用户组基本上无法使用。我们建议 收购共享蛋白质组学仪器将促进并推动我们资助的项目的成功完成 研究。所需的仪器将使我们的教师能够解决当前的技术限制 各自的领域，改善了华盛顿大学的样本分析瓶颈，极大地改善了华盛顿大学的样本分析瓶颈。 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.