Novel Hybrid Bioanalytical System

点击翻译按钮获取中文摘要

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.