High-throughput multidimensional bioseparations for next-generation proteomics

项目摘要 复杂生物样品的基于质谱（MS）的分析对于生物医学领域是必不可少的。 研究和临床诊断。分析的质量不仅取决于MS 分辨率和灵敏度，但通过用于将样品输送到仪器的液相分离， 具有更大峰容量的分离导致更多的被识别的种类和改进的 对这些身份的信任。增加分离的峰值容量目前需要 总分析时间的急剧增加，在蛋白质组覆盖率和 测量吞吐量。长的分析时间和昂贵的仪器的组合也 导致每次分析的高成本，阻碍了大量样本的研究，并强加了一个 临床诊断中常规实施的障碍。在这里，我们建议开发一个分离 方法基于液相色谱法（LC），然后快速毛细管电泳（CE）， 在较短的总分析时间内实现峰值容量分离， 降低了每个样品的成本。而不是在从LC转移到 与过去的方法一样，我们将预浓缩并聚焦从LC洗脱的样品 最近使用基于微流控阀的电动预浓缩器将柱分离成窄带 在我们的实验室里开发的。然后将聚焦的条带注入CE分离柱中， 在MS分析之前快速分离。我们将在1小时内通过CE分析约60个LC级分， 总的峰值容量接近2000人由此产生的平台应提供以下顺序 与现有方法相比，单位时间的峰值容量有了很大的提高， 这是样品测量通量、灵敏度和每次分析成本的前所未有的组合。它 也将广泛影响其他生物分析，这些分析将受益于显著改善的 通量和峰值容量，包括用于靶向蛋白质组学的选定反应监测MS， 以及代谢组学和糖组学。系统与市售产品的兼容性 LC和MS仪器将促进生物医学领域的广泛适用性和实施。 研究社区。

PROJECT SUMMARY Mass spectrometry (MS)-based analysis of complex biological samples is essential for biomedical research and clinical diagnostics. The quality of the analysis is determined not only by the MS resolution and sensitivity but by the liquid-phase separation used to deliver sample to the instrument, with separations having larger peak capacities leading to more identified species and improved confidence in those identifications. Increasing the peak capacity of a separation currently requires a dramatic increase in the total analysis time, imposing a tradeoff between proteome coverage and measurement throughput. The combination of long analysis times and expensive instrumentation also results in a high cost per analysis, impeding studies with large numbers of samples and imposing a barrier to routine implementation in clinical diagnostics. Here, we propose to develop a separation method based on liquid chromatography (LC) followed by fast capillary electrophoresis (CE) to achieve ultrahigh peak capacity separations with short overall analysis times, thus dramatically decreasing the cost per sample. Rather than waste the majority of sample during transfer from LC to CE as past approaches have done, we will preconcentrate and focus the sample eluting from the LC column into a narrow band using a microfluidic valve-based electrokinetic preconcentrator recently developed in our laboratory. The focused band will then be injected into the CE separation column for rapid separation prior to MS analysis. We will analyze approximately 60 LC fractions by CE in 1 hour, with an overall peak capacity approaching 2,000. The resulting platform should provide an order of magnitude improvement in peak capacity per unit time over existing approaches and will enable an unprecedented combination of sample measurement throughput, sensitivity and cost per analysis. It will also broadly impact other biological analyses that will benefit from dramatically improved throughput and peak capacities, including selected reaction monitoring MS for targeted proteomics, as well as metabolomics and glycomics. The compatibility of the system with commercially available LC and MS instrumentation will promote broad applicability and implementation in the biomedical research community.