Date of Award
Campus Access Thesis
Master of Science (MS)
The triacylglycerols in normal and mutant Drosophila melanogaster larvae were extracted and analyzed by reversed-phase HPLC/electrospray/tandem mass spectrometry. Ammoniated ions [M+NH4+] of the triglyceride species were formed in the electrospray process with essentially no fragmentation. The collision-induced dissociation spectra of [M+NH4+] produced diglyceride (DAG+) product ions. Mass to charge ratio of the DAG+ product ions provided fatty acid composition data and relative abundance of the DAG+ product ions provided positional information of fatty acids on the glycerol backbone.
About 30 parent ions were detected in the normal and mutant extracts. Each parent ion consisted of two to five compositional isomer systems. The degree of unsaturation for most TAG species ranged from one to three. CX:1CY:0CZ:0 was the most abundant type. Isomers co-eluted using C18 reversed phase chromatography but some isomeric species were separated using a C30 reversed phase column.
Twelve parent ions with high abundance were selected for positional analysis, including m/z 738 (C42:1), 740 (C42:0), 764 (C44:2), 766 (C44:1), 792 (C46:2), 794 (C46:1), 820 (C48:2), 822 (C48:1), 848 (C50:2), 850 (C50:1), 876 (C52:2) and 878 (C52:1). The relative intensities of the DAG product ion in the average collisional-induced decomposition spectra were used for the relative quantification of the isomers at a particular parent mass. Equations defining the relative intensities based on CID spectra of standard positional isomers were constructed and imbedded into a spreadsheet and the SOLVER function was used to fit the relative abundances of the isomers to the raw product ion intensities from the raw data. Unsaturated fatty acids were highly favored in the center position. No significant differences were seen in the relative abundances of the isomers between normal and mutant extracts for each of these twelve parent ions.
More precise measurement of the abundances of the isomeric species can be obtained using a C30 column for the separation of the TAG extracts. Separate SOLVER analyses were performed using the relative intensities of the DAG product ions in the average CID spectra across each of the resolved peaks in the ion chromatogram. The results from these separate SOLVER analyses were combined with the chromatographic peak areas to determine the relative abundances of the isomers. The uncertainties in these relative abundances were estimated by modulating the raw data input using a random number generator. The uncertainties were reduced by a factor of 2 relative to using a single SOLVER approach with the relative intensities of the DAG product ions in the average CID spectrum obtained across the whole ion chromatogram.
This thesis presented a straightforward approach for the relative quantification of TAG isomers in complex mixtures. The benefits and limitations of the technique are explored and strategies for improving the precision in future work are suggested.
Zhu, Fengying, "Analysis of Triacylglycerols in Drosophila Larvae by Reversed-Phase High Performance Liquid Chromatography-Electrospray-Tandem Mass Spectrometry and Excel-Solver" (2010). Graduate Masters Theses. Paper 12.