Mackay Merrill and Dr. John Prince, Department of Chemistry and Biochemistry
Overview
Initially, the goal of my project was to search for biologically active lipids in the lung tissue of mice with and without asthma (as opposed to those which simply form the membranes of cells) and determine if any of these lipids could serve as indicators of the disease. Over the course of experimentation, the focus shifted to these membrane (or structural) lipids. I planned to find indicative lipids (biomarkers) using mass spectrometry and statistical analysis.
Results
In order for a chemical species to be analyzed via mass spectrometry, it must have a net electrical charge. For biological species such as the lipids I studied, electrospray ionization (ESI) is often used. The Prince Lab is equipped with a nano-ESI source, an advancement of conventional ESI technology.1 I surmised that this newer technology would prove advantageous for the analysis of lipids. However, this was not the case. Achieving stable and consistent ionization with nano-ESI proved to be near impossible. Eventually, I chose to switch back to the conventional ESI source. By the time I worked out some of the major ionization problems, my samples had unfortunately degraded. Consequently, even after successful ionization of the chemical species and mass spectrometric analysis, the statistical analysis revealed no significant difference between lung tissues of mice with and without asthma.
My failure to differentiate between mice with and without asthma on the basis of their lipid content caused me to search the literature extensively for solutions.2 One monograph in particular proved especially useful3 and is now a frequent reference by all studying lipids in the Prince Lab. Among other things, this monograph confirmed my strategy to abandon nano-ESI in favor of conventional ESI technology. Eventually, I developed a consistent protocol that demonstrated stable ionization of chemical species. To confirm the validity of this protocol, I analyzed heart, liver, and lung tissue from mice to see if these could be separated on the basis of their lipid content alone. The logic behind this approach was that if these anatomically and physiologically different tissues could not be differentiated, then certainly different lung tissues (asthmatic and healthy) could not be separated. Happily, I succeeded.
Current Status
Near the end of last year, a post-doctoral researcher, Dr.Tamil Anthonymuthu, joined the Prince Lab. In consultation with Dr.Prince, it was decided that Dr. Anthonymuthu would take over my project. He has made huge strides in perfecting a protocol that will allow detailed analysis of lipids from any tissue, including new samples of lung tissue from asthmatic and healthy mice.
Moreover, my analysis of the literature proved fruitful beyond simply providing solutions to the problems I faced while studying the lipidsof asthma: I discovered LipidXplorer4, a lipid identification program; intrasource separation5, a powerful sample preparation and analysis technique; and ion fragility6, a detrimental phenomenon which significantly impacts lipid studies on Orbitrap instruments, the very type of mass spectrometer the Prince Lab uses.
In a typical shotgun lipidomics experiment, data is collected for hundreds of individual lipids. However, the identity of each lipid remains unknown. Thus, species identification is of supreme importance. LipidXplorer is designed to solve this problem and was implemented in the Prince Lab. However, my colleagues ran into problems with this software, namely consistent identification; lengthy analysis set-up; and an inability to alter the chemical ionization aid. This has led to Dr. Prince himself writing custom software which will address each of these issues. So while my discovery itself was not fruitful, it led to a specific solution to a major challenge.
Intrasource separation is a way to prepare and analyze lipid samples in a way that preferentially ionizes certain classes of lipids. The advantages of this is that instead of trying to see every lipid at the same time, targeted analyses can be performed without the use of chromatographic separation techniques. This potentially decreases signal loss of certain lipid species during analysis and allows structural information to be collected over long periods of time via fragmentation techniques. It is also a fast preparation and analysis, so many samples can be analyzed in a short amount fo time. Implementing this is the current project of my colleague, Brendan Coutu, for which he has been awarded an Undergraduate Research Award (URA) from the Department of Chemistry and Biochemistry.
Ion fragility is the phenomenon which describes the loss of a chemical species in an ion trap mass spectrometer when that species is isolated. This happens with important lipids (e.g. sphingolipids) on Orbitrap mass spectrometers, the kind used in the Prince Lab. Following the lead of a recent paper6, I am exploring how to alleviate this problem and am being funded by a URA.
Conclusion
While my initial experiment failed, it served to catalyze the creation of other projects which are currently of major importance to the Prince Lab and should produce multiple publications.
References
- Wilm, M.; Mann, M. Anal. Chem., 1996, 68 (1), 1–8
- <http://openwetware.org/wiki/Prince:BYU_Lipidomics_Team>
- Christie and Han, Lipid Analysis, 4th Edition, 2010
- Herzog, R.; et.al Genome Biology, 2011, 12, R8
- Han, X.; Gross, R. Mass Spectrom. Rev., 2005, 24 (3), 367-412
- Schumann, K.; et. al Anal. Chem., 2011, 83 (14), 5480-5487