Approach 1: direct infusion for ESI-MS/MS analysisThe crude lipid extracts are diluted in mass spectrometry-compatible solvents, directly infused into the mass spectrometer via a Trivisa NanoMate or a syringe pump or via loop injection with autosampler for analysis. The advantages of this approach are high sample throughput, relative easy operation and "looks-nice" data.
The shortcomings are
(a) lack of sensitivity for minor lipids with considerable ion suppression occurring. In most cases it is the minor lipids that are the most important signalling molecules.
(b) Considering the complexity of cellular metabolism, there is no guarantee of species selectivity if this approach is adopted.
(c) In practice this approach does not generate reliable data for many lipids, in particular PA and LPA, because of in source fragmentation, i.e., PC and PS loss head group to become PA; LPC and LPS loss head group to become LPA.
Nevertheless, it is our view that this approach can be used for high throughput analysis of the major lipids for large number samples.
Approach 2: LC-MS/MS analysisApproach 2: LC-MS/MS analysis
Lipid extracts can be separated by normal phase HPLC into different classes based upon head group polarity. ESI- MS/MS can then be used to identify the chemical structure and semi-quantify the amount of different lipid molecular species. With the aid of high resolution and accurate mass measurement, as can be achieved with the new Orbitrap Elite, high quality data is generated.
The lipid data generated using this orthogonal approach (ESI-MS/MS analysis after chromatographic separation) is much more reliable than the mass spectrometry alone direct infusion approach. The difficulty of this approach is the development of an appropriate validated HPLC method capable of clear separation of different classes of lipids as specified above.
The advantages of this approach are:
(a) very high sensitivity for trace biochemical analysis following separation of other major components in an extract.
(b) very high selectivity brought about by the separation of lipids of interest from other components which would be responsible for potential interference of the subsequent ESI-MS/MS analysis.
(c) because of in source fragmentation, it is the only way to obtain reliable data for biologically significant lipids such as PA and LPA.
Approach 3: Phosphoinositide analysisThe analysis of phosphoinositides has been complicated by the low levels of the more phosphorylated forms, their liability and the property of these highly phosphorylated forms to bind strongly to cellular proteins, glass and steel.
To address this we at Babraham have developed a method involving methylation of the phosphates, separation by reverse phase chromatography on a C4 column and neutral loss mass spectrometry (see Clark et al 2011 Nature Methods 8, 267-272). Within the facility samples are analysed by both Drs Zhang and Clark (on separate QTRAPs).
Approach 4: GC-MS/MS analysisWe established GC-MS/MS analysis of cholesterol biosynthesis intermediates such as lanosterol, 7-dehydrocholesterol, desmosterol etc nonpolar to very low polar trace lipids after silanisation of hexane extraction from Folch extract leftover from LC-MS/MS analysis of the other lipids.
Methodological Strategies in use(a) use of the direct infusion approach for major lipid analysis of large amount of samples
(b) use our unique LC-MS/MS method for analysis of important signalling lipids
(c) use of the published method for PIP2 and PIP3 analysis with potential future modification to analyse PIP and PIP2 isomers
(d) use of GC-MS/MS method for analysis of nonpolar to very low polar lipids