Novel Hybrid Bioanalytical System

描述：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，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，包括蛋白质鉴定的信心，欠采样及其定量效用。因此，测量质量通常不足以提供统计上有意义的分析，以自信地检测复杂生物医学样品中的痕量蛋白质。此外，目前大多数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，这种方法在区分生物学上重要的蛋白质异构体和鉴定蛋白质的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，用于高分辨率检测和可靠地鉴定人体体液中的完整蛋白质和蛋白质复合物，在吞吐量、灵敏度、鲁棒性和定量方面大大提高现有方法的能力。这个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，以及在目前无法检测到的浓度下对完整翻译后修饰的蛋白质和肽和蛋白质同种异构体进行定量测量的能力。先进的蛋白质组学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）和精确质量测量的高分辨率傅立叶变换质谱仪（FTMS），如Orbitrap MS，具有先进的前体离子破碎能力，包括电子转移解离（ETD）。该项目的具体目标是

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.