High Resolution Chromatography for Lipids and Proteins

在化学系化学测量和成像项目的支持下，密歇根大学的罗伯特·肯尼迪教授和他的团队正在努力改善复杂混合物的表征，如自然界和工业过程中常见的血液和土壤提取物。研究这种混合物的组成对生物学、化学、环境科学和有价值化学品的生产至关重要。分析这种混合物的一种有效方法是通过使混合物通过填充有小颗粒（“固定相”）的色谱柱来将化学组分彼此分离，所述小颗粒与不同组分在不同程度上相互作用。当化学成分从柱子中出来时，它们被一种称为“质谱仪”的设备进行分析，以进行鉴定和定量。分离混合物组分的能力可以通过使用更小的颗粒和更长的柱来提高，但是这样的变化需要更高的压力来驱动样品通过柱。目前的技术在很大程度上限于在低于20，000 psi的压力下操作。因此，许多混合物不能被正确地分析。肯尼迪集团正在努力通过开发可以在高达100，000 psi的压力下运行的色谱系统来缓解这一限制。改进后的色谱法将使混合物在许多学科中得到更好的表征。例如，该小组正在使用新方法来识别与能够有效生产生物燃料的微生物相关的生物分子模式（主要是脂质和蛋白质）。 努力传播有关重要方法、原理和应用的知识，包括本科生的参与和对更年轻学生的推广，包括代表性不足的群体的成员。分析分离对复杂混合物分析至关重要。高压液相色谱法（HPLC）是一种主要的分离方法。 为了分析许多复杂的混合物，有必要实现比目前可能的更高的分辨率。 Kennedy集团正在开发用于化学分析的下一代高分辨率分离技术。他们正在通过开发a）可常规使用高达100 kpsi的配件; B）长柱中小颗粒（0.5至1.7 μ m）的填充条件;以及c）长柱中不同LC模式（如亲水相互作用色谱（HILIC）和离子交换（IEX））的填充条件来推进超高压液相色谱（UHPLC）。他们还将其新型UHPLC色谱柱与其他分离方法相结合，用于二维（2D）分离和质谱（MS）。他们的目标是通过脂质和完整蛋白质的2D色谱实现前所未有的峰容量（10，000），期望增加峰容量将导致以更好的灵敏度和重现性检测复杂样品中的更多化合物。在一个具体的应用中，他们正在探索细菌中的脂质组和蛋白质组重排，这些细菌对燃料产品的毒性产生了抵抗力。鉴定这种重排对于工程细菌用于高产生物燃料生产至关重要。 这项工作正在通过与国家高磁场实验室的合作者的互动而广泛提供。该奖项反映了NSF的法定使命，并被认为值得通过使用基金会的智力价值和更广泛的影响审查标准进行评估来支持。

With support from the Chemical Measurement and Imaging Program in the Division of Chemistry, Professor Robert Kennedy and his group at the University of Michigan are working to improve the characterization of complex mixtures such as blood and soil extracts, common in nature and industrial processes. Studying the composition of such mixtures is vital to biology, chemistry, environmental science, and production of valuable chemicals. A powerful approach to analyzing such mixtures begins by separating chemical components from one another by passing the mixture through a chromatographic column packed with small particles ("stationary phase") that interact to varying degrees with different components. As chemical components emerge from the column they are analyzed by a device called a "mass spectrometer" for identification and quantification. The ability to separate mixture components can be improved by using smaller particles and longer columns, but such changes require higher pressure to drive the sample through the column. Current technology is largely limited to operating at pressures below 20,000 psi. As a result, many mixtures cannot be properly analyzed. The Kennedy group is working to relieve this constraint by developing chromatographic systems that can operate at up to 100,000 psi. The improved chromatography will allow better characterization of mixtures in many disciplines. For example, the group is using the new methods to identify biomolecular patterns (primarily lipids and proteins) associated with microbes capable of efficient biofuel production. Efforts to disseminate knowledge about the important methods, principles, and applications include involvement of undergraduate students and outreach to even younger students, including members of underrepresented groups.Analytical separations are critical to complex mixture analysis. High-pressure liquid chromatography (HPLC) is a dominant method for separations. To analyze many complex mixtures, it is necessary to achieve higher resolution than what is currently possible. The Kennedy group is developing next-generation, high-resolution separations for chemical analysis. They are advancing ultra-high pressure liquid chromatography (UHPLC) through development of a) fittings that can be routinely used up to 100 kpsi; b) packing conditions for small particles (0.5 to 1.7 um) in long columns; and c) packing conditions for different modes of LC, such as hydrophilic interaction chromatography (HILIC) and ion-exchange (IEX), in long columns. They are also coupling their new UHPLC columns with other separation methods in two-dimensional (2D) separations coupled to mass spectrometry (MS). They are targeting unprecedented peak capacity (10,000) by 2D chromatography of lipids and intact proteins, with the expectation that increasing peak capacity will result in detection of more compounds in complex samples with better sensitivity and reproducibility. In a specific application, they are probing the lipidome and proteome rearrangements in bacteria that develop resistance to toxicity of fuel products. Identification of such rearrangements is crucial for engineering bacteria for high-yield biofuel production. The work is being made broadly available through interactions with collaborators at the National High Magnetic Field Laboratory.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.