Multidimensional proteomics platform

描述(申请人提供)：尽管新的蛋白质生物标记物的开发前景令人兴奋，但目前的蛋白质组测量能力在关键方面缺乏，如吞吐量、低丰度蛋白质的检测极限、数据质量(包括蛋白质鉴定和欠采样的置信度)和定量准确性。结果，测量质量通常不足以在广泛的生物医学分析中自信地检测微量蛋白质，并且可能的吞吐量不允许对大多数目的所需的分析进行统计。其结果是，在生物医学样本的广泛分析中，测量质量通常不足以自信地检测微量蛋白质，并且可能的吞吐量不足以为大多数目的提供具有统计意义的分析数量。此外，目前的大多数蛋白质组学平台最适合于检测蛋白水解肽，使用的是“自下而上”的方法，这种方法在区分具有生物学意义的重要蛋白质异构体和鉴定蛋白质翻译后修饰方面效率低下。该项目的总体目标是开发一个应用于人体体液的候选蛋白质生物标记物发现和验证分析平台，该平台将在吞吐量、灵敏度、稳健性和定量方面大大改进现有的方法。新平台旨在提供更大的蛋白质组覆盖范围，并能够在目前存在问题的浓度下对完整蛋白质和较高分子量肽进行定量测量。该平台将包括快速毛细管区带电泳(CZE)分离，集成了微流控芯片上的新型微制造纳米电喷雾电离(Nano-ESI)发射器，然后是连接到高分辨率气相离子迁移率(IMS)分离级的低压场非对称离子迁移谱仪(FAIMS)和高分辨率飞行时间质谱仪(TOF MS)，提供精确的质量测量。本项目的具体目标是：(1)设计一种结合了CZE和纳米ESI源的一次性微流控芯片；(2)在完整蛋白质的分析中，建立并开发一种可将化学背景水平降至最低的减压FAIMS设备；(3)开发一种结合气相IMS分离的高效离子激活方法；以及(4)通过临床样本对多维蛋白质组学平台进行严格的评估。我们预计，拟议的蛋白质组学平台将提供整体高灵敏度和分离能力，以及准确的定量，以更有效和更高吞吐量的测量来发现生物液中识别生物状态的蛋白质、蛋白质片段和多肽的分析模式。 公共卫生相关性(由申请人提供)：该项目的具体目标是开发一个高通量、灵敏的多维分离平台，用于定量分析人体体液中完整的蛋白质和较高分子量肽。该平台将在一次性微流控芯片上使用毛细管区带电泳和纳米电喷雾电离源实现浓缩相蛋白质的分离和电离，然后是高效的气相离子迁移率分离和质谱仪检测。

DESCRIPTION (provided by applicant): While the development of new protein biomarkers is an exciting prospect, current proteomic measurement capabilities are lacking in key aspects, such as throughput, detection limits for low-abundance proteins, and data quality (including the confidence of protein identifications and under-sampling), and quantitation accuracy. In the result, the measurement quality is generally insufficient to confidently detect trace proteins in broad biomedical analyses, and the throughput possible does not allow the statistics of analyses needed for most purposes. The result is that measurement quality is generally insufficient to confidently detect trace proteins in broad analyses of biomedical samples, and the throughput possible is insufficient to provide statistically meaningful numbers of analyses for most purposes. In addition, most of the current proteomics platforms are best suited for detection of proteolytic peptides, using the "bottom-up" approach which is inefficient for distinguishing between biologically important protein isoforms and for identifications of post translational modifications of proteins. The overall objective of this project is to develop a platform for candidate protein biomarker discovery and verification analyses for application to human bodily fluids that will greatly improve upon existing methodologies in terms of throughput, sensitivity, robustness, and quantitation. The new platform aims at providing greater proteome coverage and enabling quantitative measurements of intact proteins and higher molecular weight peptides at concentrations that are presently problematic. The platform will encompass fast capillary zone electrophoresis (CZE) separation integrated with a novel microfabricated nano-electrospray ionization (nano-ESI) emitter on a microfluidic chip followed by a lower pressure field asymmetric ion mobility spectrometer (FAIMS) interfaced to a high resolution gas phase ion mobility (IMS) separation stage and a high-resolution time-of-flight mass spectrometer (TOF MS), providing accurate mass measurements. The specific aims of this project are to: (1) design a single-use disposable microfluidic chip which incorporates CZE and nano-ESI source, (2) model and develop a reduced pressure FAIMS device for minimizing chemical background levels in analysis of intact proteins, (3) develop an efficient ion activation approach in conjunction with gas-phase IMS separation, and (4) rigorously evaluate the multidimensional proteomics platform with clinical samples. We anticipate that the proposed proteomics platform will provide the overall high sensitivity and separation power, as well as accurate quantitation, required for more effective and higher throughput measurements to discover assay patterns of proteins, protein fragments, and peptides in biological fluids that identify biological states. PUBLIC HEALTH RELEVANCE (provided by applicant): The specific objective of this project is to develop a high-throughput sensitive multidimensional separation platform for quantitative analysis of intact proteins and higher molecular weight peptides from human bodily fluids. The platform will enable condensed-phase protein separations and ionization using capillary zone electrophoresis and nano-electrospray ionization source on a single-use disposable microfluidic chip followed by efficient gas-phase ion mobility separations and mass spectrometry detection.