Enhancing Coverage of Phosphatidylinositol Species in Canola Through Specialised Liquid Chromatography-Mass...

Phosphatidylinositols (PIs) constitute a minor class of phospholipid with wide-spread influence throughout various cellular functions. Monitoring the distribution of these lipids can therefore provide insight as to the state of cellular processes or reveal the development of various pathologies. The speciation of these compounds is often performed either as part of a comprehensive characterisation of lipids, or specifically targeted using the same methods, however, such methods were intended to maximise coverage of lipid classes rather than provide an in-depth analysis of any single class. In the particular case of PIs, the majority of reported molecular diversity is limited to a small proportion of the already minor class, as such the cursory glance enabled by such methods is insufficient. Therefore, this work compared the suitability of both established and novel LC-MS buffers with the aim of maximising the ionisation efficiency of PIs, in an attempt to enhance coverage of the class. Through experimentation, it was determined that a 0.25 mM ammonium fluoride buffer provided up to a 6-fold increase in signal intensity, and on average a 38-fold increase in the signal-to-noise ratio. Using these new conditions, 14 PI species, and 12 PI candidates were identified within a dilute lipid extract sourced from canola seed, compared to 0 species identified using the generalised method. As a result, it is suggested that this procedure has yielded the highest number of PI species identifications for a sample of this concentration. Methods which therefore intend to characterise PI species in dilute quantities, such as those extracted from mammalian cells, are henceforth provided with the means to conduct more comprehensive characterisations.