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
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.