A PROTEOMICS RESEARCH RESOURCE FOR INTEGRATIVE BIOLOGY

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。本年度报告的具体目标1侧重于与自动化分析、蛋白质组样品处理相关的发展，以及现场不对称波形离子迁移率光谱分析仪的新发展。自动化高通量高效液相色谱高通量毛细管高效液相色谱系统已在公司内部成功设计和开发，目前正用于蛋白质组样品的常规分析。该系统基于我们实验室之前开发的一种设计的流体成分加倍，该设计允许通过应用一种新的技术将柱与基于编码翻译阶段的质谱仪接口，从而重叠四个分离柱的处理。与我们实验室开发的原始单柱设计相比，4柱系统具有许多优势，包括吞吐量、成本和尺寸。首先，与单列系统相比，4列系统具有~100%的占空比，并且没有伴随的数据质量损失。在24小时内，4柱系统具有~14个样品的吞吐量，而1柱系统仅有~7个样品。其次，这种样品吞吐量的增加可以通过升级现有的单柱系统来实现，与购买另一个单柱系统和质谱仪的替代方案相比，可以节省大量成本。最后，升级现有系统不会增加LC/MS系统(S)实现吞吐量增加所需的占用空间，而购买新系统会使其增加一倍。可在Beckman Biomek FX自动液体处理机器人上执行的协议的开发和改进正在进行中。与德斯蒙德·史密斯合作的NCRR项目对自动化样本处理的开发和改进做出了重大贡献，因为与该项目相关的大量小样本-小鼠脑组织片段被称为脑体素。单个小鼠大脑可以产生~400-600个L体素，正在开发在96孔板中处理它们的方法。从每个体素中提取的蛋白质使用为Biomek FX开发的程序进行胰酶消化，以处理溶液中的蛋白质组样本(即，不是在2D-PAGE凝胶中分离)。该项目的未来工作将包括自动化脑体素样品处理协议的更多步骤，以及将准备用于MS分析的样品以96孔板的形式传输到LC-MS系统(目前LC-MS系统仅针对单个样品瓶进行配置)。PNNL的平面FAIMS开发之前，我们报告了场不对称波形离子迁移率谱(FAIMS)分析仪的先验模拟软件的开发，在2005年，我们使用该软件包作为间隙曲率的函数来优化FAIMS的性能，特别是比较平面(p-)和圆柱(c-)几何形状。计算表明，p-FAIMS具有最高的分辨率，比具有典型曲率的c-FAIMS高2-5倍。这一改进适用于任何类型的离子，并在较高的绝对补偿电压(CV)时增加。在建模的指导下，我们建立了一个新的高分辨率p-FAIMS单元并进行了评估。与仿真结果一致，分辨率增益最高可达300%，并随CV的增大而增大。这种性能使得以前FAIMS不可能实现困难的异构体分离，例如区分(I)质子化亮氨酸和异亮氨酸，以及(Ii)至少四个H+缓激肽构象，而不是使用c-FAIMS发现的两个。蛋白质构象的分离也有所改善，尽管幅度较小(可能是因为构象集合固有的多样性)。新的p-FAIMS还具有集成的ESI接口，提供有效的离子去溶，使该系统适用于蛋白质组和其他生物样本的有效分析。仿真还表明，在分辨率/灵敏度平衡方面，p-FAIMS优于c-FAIMS，即在相同灵敏度下获得更高的分辨率或在相同分辨率下获得更高的灵敏度。然而，由于缺乏有效的MS接口，p-FAIMS的广泛采用受到阻碍。我们已经开发和展示了一种新的狭缝孔径MS(和IMS)界面，与电动离子漏斗相结合，可以更好地捕获来自p-FAIMS的漫射带状离子束，从而提高FAIMS/MS分析的灵敏度。进一步提高p-FAIMS/MS结离子利用率的工作正在进行中。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Specific Aim 1 for this annual report focuses on developments associated with automated analysis, proteome sample processing, and new developments with field asymmetric waveform ion mobility spectrometry analyzers. Automated High-Throughput Liquid Chromatography A high-throughput capillary HPLC system has been successfully designed and developed in-house and is now being used for routine analysis of proteomic samples. This system is based on ¿doubling¿ the fluidic components of a design previously developed in our laboratory which allows it to overlap the processing of four separation columns by applying a novel technique for interfacing the columns with a mass spectrometer based on encoding translation stages. The 4-column system has a number of advantages over the original 1-column design developed in our laboratory including throughput, cost, and size. First, the 4-column system has a duty cycle of ~100% with no accompanying loss in data quality compared to the 1-column system. In 24 hours the 4-column system has a throughput of ~14 samples compared to only ~7 samples for the 1-column system. Second, this increase in sample throughput can be accomplished by ¿upgrading¿ an existing 1-column system at a substantial savings compared to the alternative of acquiring another 1-column system and a mass spectrometer. Finally, upgrading an existing system does not increase the footprint of the LC/MS system(s) needed to achieve the increase in throughput while acquiring a new system doubles it. Automated Sample Handling Development and refinement of protocols that can be performed on a Beckman Biomek FX automated liquid-handling robot are in progress. The NCRR collaborative project with Desmond Smith has contributed significantly to the development and refinement of automated sample handling due to the extensive numbers of small samples associated with this project - pieces of mouse brain tissue referred to as brain ¿voxels.¿ A single mouse brain can yield ~400-600 1 ¿l voxels and methods are being developed to process them in 96-well plates. The proteins that are extracted from each voxel are tryptically digested using a protocol developed for the Biomek FX for processing proteome samples in solution (i.e., not isolated in a 2D-PAGE gel). Future work on this project will involve automating more steps of the brain voxel sample-handling protocol in addition to transferring samples ready for MS analysis to an LC-MS system in a 96-well plate format (currently the LC-MS systems are configured only for individual sample vials). Planar FAIMS Development at PNNL Having previously reported the development of software for a priori simulation of Field Asymmetric waveform Ion Mobility Spectrometry (FAIMS) analyzers, in 2005 we used the package to optimize FAIMS performance as a function of gap curvature, in particular, comparing planar (p-) and cylindrical (c-) geometries. Calculations revealed that p-FAIMS offers the maximum resolution, exceeding that of c-FAIMS with typical curvatures by ~2 - 5 times. The improvement applies to ions of any type and increases at higher absolute compensation voltages (CV). Guided by modeling, we have built and evaluated a new high-resolution p-FAIMS unit. In agreement with simulations, the resolution gain is up to 300%, increasing with increasing CV. That performance has enabled difficult isomeric separations not possible by FAIMS previously, such as distinguishing (i) protonated Leucine and Isoleucine and (ii) at least four H+bradykinin conformers vs. two found using c-FAIMS. Separation of protein conformers has also improved, though by a smaller margin (likely because of inherent diversity of conformer ensembles). The new p-FAIMS also features an integrated ESI interface providing effective ion desolvation, making the system suitable for effective analysis of proteomic and other biological samples. Simulations also show the superiority of p- over c- FAIMS in terms of resolution/ sensitivity balance, meaning a higher resolution at equal sensitivity or higher sensitivity at equal resolution. However, broad adoption of p-FAIMS was impeded by lack of effective MS interfaces. We have developed and demonstrated a new slit-aperture MS (and IMS) interface that, in conjunction with an electrodynamic ion funnel, provides a better capture of diffuse ribbon-shaped ion beams exiting p-FAIMS and thus improves the sensitivity of FAIMS/MS analyses. Efforts to increase the ion utilization at the p-FAIMS/MS junction further are in progress.