Novel Hybrid Bioanalytical System

产品说明：虽然完整蛋白质和蛋白质复合物的检测和表征是一个令人兴奋的前景，但目前的蛋白质组学测量能力在各种关键方面都缺乏，例如测量通量，低丰度蛋白质的检测，以及与数据质量相关的实质性问题，包括蛋白质鉴定的置信度，欠采样及其定量效用。因此，测量质量通常不足以提供有统计学意义的分析，以自信地检测复杂生物医学样品中的痕量蛋白质。此外，大多数目前的蛋白质组学平台最适合于检测蛋白水解肽，使用“自下而上”的方法，这对于区分生物学上重要的蛋白质同种型和鉴定蛋白质的翻译后修饰是低效的。 本项目的总体目标是开发一种集成系统，用于高分辨率检测和可靠鉴定人体体液中的完整蛋白质和蛋白质复合物，在通量、灵敏度、鲁棒性和定量方面大大提高了现有方法的能力。该新系统旨在提供完整蛋白质和肽的翻译后修饰的可靠定位以及在目前不可检测的浓度下定量测量完整翻译后修饰的蛋白质和肽以及蛋白质同种型的能力。一个先进的蛋白质组学系统将基于双离子源，包括纳米电喷雾离子化（纳米-ESI）和表面声波雾化（SAWN）与高分辨率气相离子迁移谱仪（IMS）和精确质量测量高分辨率傅里叶变换质谱仪（FTMS）（诸如具有先进前体离子碎裂能力的Orbitrap MS）接口，包括电子转移解离（ETD）。该项目的具体目标是 1）开发高分辨率高灵敏度漂移管离子迁移谱仪（IMS），用于分析较大的肽、蛋白质片段和完整蛋白质。在不低于80%的总离子透射率下实现大于150的IMS分辨率，2）使用具有双重掩蔽的先进多路复用方法将配备有纳米电喷雾电离（ESI）源的高分辨率IMS仪器连接到Orbitrap MS，并在大于100的质量分辨率下实现完整IMS-Orbitrap系统的不低于30%的占空比，3）将表面声波雾化（SAWN）装置连接到高分辨率IMS-Orbitrap MS仪器，并在人血清/血浆样品的实验中验证配备有SAWN和纳米-ESI源的IMS-Orbitrap MS系统的性能。演示使用新型nano-ESI（SAWN）-IMS-Orbitrap仪器分析分离蛋白质异构体、后修饰肽/蛋白质变体和非共价结合蛋白质复合物。 我们预期新型ESI（SAWN）-IMS-Orbitrap仪器的开发将提供最高质量分辨率和质量准确度的无与伦比的能力，具有整体高灵敏度和IMS分离能力，以及生物流体中的后修饰蛋白质、蛋白质同种型和蛋白质复合物的更有效和更高通量测量所需的准确定量。

DESCRIPTION: While the detection and characterization of intact proteins and protein complexes is an exciting prospect, current proteomics measurement capabilities are lacking in a variety of crucial aspects such as measurement throughput, detection of low abundance proteins, and substantial issues associated with data quality including the confidence of protein identifications, under-sampling and their quantitative utility. Consequently, the measurement quality is generally insufficient to provide statistically meaningful analyses to confidently detec trace proteins within complex biomedical samples. 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 proect is to develop an integrated system for high- resolution detection and reliable identification of intact proteins and protein complexes from human bodily fluids with greatly improved capabilities over the existing methodologies in terms of throughput, sensitivity, robustness, and quantitation. This new system aims at providing reliable localization of post-translational modifications of intact proteins and peptides and the ability for quantitative measurements of intact post- translationally modified proteins and peptides and protein isoforms at concentrations that are presently undetectable. An advanced proteomic system will be based upon a dual ion source encompassing both nano-Electrospray Ionization (nano-ESI) and Surface Acoustic Wave Nebulization (SAWN) interfaced to a high resolution gas phase ion mobility spectrometer (IMS) and an accurate-mass-measurement high-resolution Fourier Transform Mass Spectrometer (FTMS), such as an Orbitrap MS, with advanced precursor ion fragmentation capabilities, including electron transfer dissociation (ETD). The specific aims of this project are to 1) Develop a high-resolution high-sensitivity drift- tube Ion Mobility Spectrometer (IMS) for analysis of larger peptides, protein fragments and intact proteins. Achieve IMS resolution of greater than 150 at an overall ion transmission of no less than 80 %, 2) Interface the high-resolution IMS instrument equipped with a nano-electrospray ionization (ESI) source to an Orbitrap MS using an advanced multiplexed approach with double masking and attain the duty cycle of no less than 30% for the complete IMS-Orbitrap system at a mass resolution of greater than 100,000 and a mass accuracy of < 3 ppm in the analysis of complex protein mixtures and 3) Interface a Surface Acoustic Wave Nebulization (SAWN) device to high- resolution IMS-Orbitrap MS instrument and validate performance of the IMS-Orbitrap MS system equipped with both SAWN and nano-ESI sources in experiments with human blood serum/plasma samples. Demonstrate analytical separation of protein isoforms, variants of post-translationally modified peptides/proteins and non-covalently bound protein complexes with the novel nano-ESI (SAWN)-IMS-Orbitrap instrumentation. We anticipate that the development of the novel ESI (SAWN)-IMS-Orbitrap instrument will provide unparallel capabilities of highest mass resolution and mass accuracy with the overall high sensitivity and IMS separation power, as well as accurate quantitation required for more effective and higher throughput measurements of post- translationally modified proteins, protein isoforms, and protein complexes in biological fluids.