Novel Hybrid Bioanalytical System

描述：虽然完整蛋白质和蛋白质复合体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许多关键方面缺乏，如测量吞吐量、低丰度蛋白质的检测，以及与数据质量相关的实质性问题，包括蛋白质识别的可信度、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意义的分析，以自信地检测复杂生物医学样本中的微量蛋白质。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种集成系统，用于从人体体液中高分辨率检测和可靠地识别完整的蛋白质和蛋白质复合体，在吞吐量、灵敏度、稳健性和定量方面比现有方法有很大的改进。这一新系统旨在提供完整蛋白质和多肽的翻译后修饰的可靠定位，以及在目前无法检测到的浓度下定量测量完整的翻译后修饰的蛋白质和多肽和蛋白质异构体的能力。先进的蛋白质组系统将基于双离子源，包括纳米电喷雾电离(Nano-ESI)和表面声波雾化(SAWN)，与高分辨率气相离子迁移谱仪(IMS)和具有先进的前体离子裂解能力(包括电子转移解离(ETD))的精确质量测量高分辨率傅立叶变换质谱仪(FTMS)相连接。这个项目的具体目标是 1)研制高分辨率高灵敏度漂移管离子迁移谱仪(IMS)，用于分析较大的多肽、蛋白质片段和完整蛋白质。在总离子透过率不低于80%的情况下获得大于150的IMS分辨率，2)将配备纳米电喷雾电离(ESI)源的高分辨率IMS仪器与采用先进的双屏蔽多路复用方法的Orbitrap MS接口，在质量分辨率大于100,000和质量精度&lt的情况下，对于整个IMS-Orbitrap系统，获得不低于30%的占空比；3)将声表面波雾化(SAWN)装置连接到高分辨率的IMS-Orbitrap MS仪器，并在人血清/血浆样品的实验中验证同时配备SAWN和纳米ESI源的IMS-Orbitrap MS系统的性能。使用新型纳米ESI(SAWN)-IMS-Orbitrap仪器演示蛋白质异构体、翻译后修饰多肽/蛋白质变体和非共价结合蛋白质复合体的分析分离。我们预计，新型ESI(SAWN)-IMS-Orbitrap仪器的开发将提供无与伦比的最高质量分辨率和质量精度能力，以及整体高灵敏度和IMS分离能力，以及更有效和更高吞吐量测量生物体液中翻译后修饰蛋白质、蛋白质异构体和蛋白质复合体所需的准确定量。

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.