High-throughput multidimensional bioseparations for next-generation proteomics

项目总结 基于质谱学的复杂生物样品分析是生物医学研究的重要内容 研究和临床诊断学。分析的质量不仅由MS决定 分辨率和灵敏度，而是通过用于将样品输送到仪器的液相分离， 具有更大的峰容量的分离导致更多的鉴定物种和改进 对这些身份的信心。增加分选的峰值容量目前需要 极大地增加了总分析时间，在蛋白质组覆盖和 测量吞吐量。分析时间长和昂贵的仪器设备的组合也 导致每次分析的成本很高，阻碍了大量样本的研究，并迫使 临床诊断中常规实施的障碍。在这里，我们建议开发一种分离 高效毛细管电泳联用快速毛细管电泳法 以较短的整体分析时间实现超高的峰容量分离，从而显著 降低每件样品的成本。而不是在从LC转移到 正如过去的方法一样，我们将对从LC洗脱出来的样品进行预浓缩和聚焦 最近使用基于微流体阀的电动预浓缩器将柱子变成窄带 这是我们实验室研发的。聚焦后的波段将被注入CE分离柱，用于 在MS分析之前进行快速分离。我们将在1小时内用毛细管电泳法分析大约60个LC组分， 整体高峰运力接近2,000辆。生成的平台应该提供一个顺序为 与现有方法相比，单位时间的峰值容量大幅提高，并将使 前所未有的样品测量吞吐量、灵敏度和每次分析成本的组合。它 也将广泛影响其他生物分析，这些分析将受益于显著的改进 吞吐量和峰容量，包括针对靶向蛋白质组的选定反应监测MS， 以及代谢组学和糖组学。该系统与商业上可获得的系统的兼容性 LC和MS仪器将促进生物医学的广泛应用和实施 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.