High Resolution Chromatography for Lipids and Proteins

Regents Of The University Of Michigan - Ann Arbor, Ann Arbor MI

Investigators

Abstract

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.