High Resolution Chromatography for Lipids and Proteins

在化学系化学测量和成像计划的支持下，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血液和土壤提取物等复杂混合物的特征，这些混合物在自然界和工业过程中很常见。研究这种混合物的组成对生物、化学、环境科学和生产有价值的化学品至关重要。分析这种混合物的一种强有力的方法是将化学成分相互分离，将混合物通过装满小颗粒(固定相)的色谱柱，这些小颗粒与不同的成分有不同程度的相互作用。当化学成分从色谱柱中出现时，它们会被一种名为“质谱仪”的设备进行分析，以进行鉴定和定量。使用更小的颗粒和更长的色谱柱可以提高分离混合物组分的能力，但这种变化需要更高的压力来推动样品通过色谱柱。目前的技术在很大程度上仅限于在20000磅/平方英寸以下的压力下运行。因此，许多混合物不能被正确地分析。肯尼迪团队正在努力通过开发可以高达10万磅/平方英寸的色层分析系统来缓解这一限制。改进的层析将允许在许多学科中更好地表征混合物。例如，该小组正在使用新方法来识别与能够高效生产生物燃料的微生物相关的生物分子模式(主要是脂类和蛋白质)。传播有关重要方法、原理和应用的知识的努力包括本科生的参与和更年轻的学生的参与，包括代表人数不足的群体的成员。分析分离是复杂混合物分析的关键。高压液相色谱(HPLC)是主要的分离方法。要分析许多复杂的混合物，有必要达到比目前可能的分辨率更高的分辨率。肯尼迪团队正在开发用于化学分析的下一代高分辨率分离器。他们正在通过开发a)可常规使用高达100 kpsi的配件；b)在长柱中填充小颗粒(0.5至1.7微米)的条件；以及c)在长柱中填充不同模式的LC，例如亲水相互作用层析(HILIC)和离子交换(IEX)，从而推动超高压液相色谱(UHPLC)的发展。他们还将其新的超高效液相色谱柱与其他分离方法在二维(2D)分离和质谱分析(MS)中进行耦合。他们的目标是通过脂类和完整蛋白质的2D层析获得前所未有的峰容量(10,000)，期望增加峰容量将以更好的灵敏度和重复性在复杂样品中检测到更多化合物。在一个特定的应用中，他们正在探索细菌中对燃料产品毒性产生抵抗力的脂体和蛋白质组重排。鉴定这种重排对于高产生物燃料生产的工程菌至关重要。这项工作正通过与国家强磁场实验室的合作者的互动广泛提供。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

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.