Multidimensional proteomics platform

多维蛋白质组学平台

• 批准号: 8132940
• 负责人: Yehia Ibrahim
• 金额: $ 19.84万
• 依托单位: BATTELLE PACIFIC NORTHWEST LABORATORIES
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-01 至 2013-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8132940
• 关键词: Biological; Biological Assay; Biological Markers; Blood capillaries; Capillary Electrophoresis; Chemicals; Clinical; Data; Data Quality; Detection; Development; Devices; Electrophoresis; Electrospray Ionization; Electrostatics; Ensure; Excision; Financial compensation; Frequencies; Gases; Heating; Human; Ions; Liquid substance; Mass Spectrum Analysis; Measurement; Methodology; Microfluidics; Modeling; Molecular Weight; Motion; Pattern; Peptides; Performance; Phase; Post-Translational Protein Processing; Protein Fragment; Protein Isoforms; Proteins; Proteome; Proteomics; Radio; Resolution; Sampling; Scanning; Source; Staging; Time; base; capillary; design; improved; instrument; ion mobility; ionization; mass spectrometer; model design; nano-electrospray; novel; novel strategies; pressure; protein complex; public health relevance; statistics; voltage

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.

描述（由申请人提供）：虽然新蛋白质生物标志物的开发是一个令人兴奋的前景，但是当前的蛋白质组学测量能力在关键方面缺乏，例如吞吐量，低育蛋白的检测限和数据质量（包括蛋白质识别和透露率识别和不足的置信度），以及准确度。在结果中，测量质量通常不足以在广泛的生物医学分析中有信心检测痕量蛋白，并且可能的吞吐量不允许大多数目的所需的分析统计数据。结果是，在对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痕量蛋白，并且可能的吞吐量不足以为大多数目的提供统计学上有意义的分析。此外，使用“自下而上”的方法，当前大多数当前的蛋白质组学平台最适合于检测蛋白水解肽，该方法无法区分生物学上重要的蛋白质同工型和鉴定蛋白质后翻译的鉴定。该项目的总体目的是开发一个平台，用于候选蛋白质生物标志物发现和验证分析，以应用于人体流体，这将在吞吐量，敏感性，稳健性和定量方面大大改善现有方法。新平台旨在提供更大的蛋白质覆盖范围，并在目前有问题的浓度下对完整蛋白质和更高的分子量肽进行定量测量。该平台将在微流体芯片上与新型的微生物纳米纳米电离电离（Nano-ESI）相结合的快速毛细管电泳（CZE）分离，然后在微流体芯片上发射器，然后是较低的压力场离子迁移率计（FAIMS）与高分辨率分离型（IMS-IOM-IOM IOM-ASION IOM ASSION IOM-ION-ION IOM-FERIPATION and SS-IN-IN-IN-IN-IN-IN-IMS-IN-IN-IMS-IN-IN-IMS-IN-IN-IMS-IN-IN-IMS-IMS-IIN-IN-IMS-IN-IIN-IIN-IN-IN-IN-IMS）。光谱仪（TOF MS），提供准确的质量测量值。该项目的具体目的是：（1）设计一种一次性的一次性微流体芯片，该芯片结合了CZE和Nano-ESI来源，（2）模型并开发了降低的压力FAIMS设备，以最大程度地减少对完整蛋白质分析的化学背景水平，（3）与CONJUNTION一起评估了多个ION激活方法，并将其与Gas-iim分离，并（4）和（4）IMSS和4（4）IMS（4）具有临床样本的蛋白质组学平台。我们预计，提出的蛋白质组学平台将提供总体高灵敏度和分离能力以及准确的定量，这是更有效和更高的吞吐量测量所需的，以发现鉴定生物学状态的生物学流体中蛋白质，蛋白质碎片和肽的测定模式。 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敏感的多维分离平台，以定量完整蛋白质和人体体液中较高的分子量肽的定量分析。该平台将使用毛细管区电泳和纳米 - 电喷雾电离源在单使用一次性的微流体芯片上，然后进行有效的气相离子迁移率分离和质谱检测。