Development of a shotgun lipidomics approach for analysis of lipids in perennial ryegrass : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Chemistry at Massey University, Manawatu, New Zealand

Abstract

Mass Spectrometry is a powerful analytical tool which is used for identification and quantitation of compounds within samples for a variety of sample matrices. One example of this is to look into the lipid profile (lipidome) of perennial ryegrass (lolium perenne). The lipid profile tells us many things about the inner workings of rye grass which can lead to better understanding of mechanisms behind desirable traits (such as lipid quantity and composition). Traditional high performance liquid chromatography (HPLC) is the most widely used chromatographic technique when researching into the lipidome of different plants (Burgos et al., 2011; Chen, Markham, & Cahoon, 2012; Degenkolbe et al., 2012). Shotgun lipidomics applies major principles of the traditional methods but differs in the delivery of the sample to the Mass Spectrometer and data analysis; providing considerable advantages, disadvantages and challenges. A shotgun lipidomics method for analysing the lipids in perennial ryegrass has been developed. This involved first determining the most efficient extraction protocol and then establishing a methodology (based on one found in the literature for animal samples) for shotgun lipidomic analysis of perennial ryegrass. The shotgun data was problematic to analyse using traditional methods so LCMS data was investigated and the results were transferred to the shotgun data. Investigation was conducted to find the limiting factor for the analysis of the shotgun data. This limiting factor was found to be pheophytin a and other chlorophyll derivatives. The high abundance and ion suppression effects attributed to pheophytin a and other chlorophyll derivatives contributed to unfavourable conditions for analysing the lipidome of perennial ryegrass. The major outcomes of this study are the annotation of 118 lipids in perennial ryegrass using LCMS, with 27 of those being found in the shotgun data also and also the understanding of the limitations of using shotgun techniques for perennial ryegrass. With this understanding further research can be conducted to enhance the methodologies detailed herein.