br Experimental Procedures br Author Contributions br Acknow
Introduction Oxysterols came to prominence in the late 1970's with the oxysterol hypothesis which proposed that the suppressive effect of cholesterol on its own synthesis is mediated through oxysterols not by cholesterol itself . This has proved to be only partly true, as cholesterol homeostasis in calcifediol is modulated through oxysterols, side-chain oxysterols inhibiting the SREBP2 (sterol regulatory element-binding protein 2) pathway and also activating liver X receptors (LXRs) [2,3], although cholesterol itself is in fact the major regulator of its own synthesis through binding to SCAP (SREBP cleavage activating protein) and preventing transport of SERBP2 from the endoplasmic reticulum for processing to its active form as a transcription factor for genes of the cholesterol biosynthesis pathway . In recent years oxysterols have been shown to have important functions in immunology [, , , , , , , , ], development [14,15] and cancer [, , , , ]. This has stimulated wide-spread interest in their analysis using lipidomics technology [21,22]. Although mostly thought of as oxidised forms of cholesterols, oxysterols can also be formed from precursors of cholesterol greatly widening the range of molecules required to be analysed in a lipidomic study.
Mass spectrometry-based technologies Oxysterols tend not to be observed in global lipidomic analysis, whether shot-gun based electrospray ionisation – mass spectrometry (ESI-MS) or liquid chromatography (LC)-MS based. This is because of their comparatively low-abundance and poor ionisation characteristics. However, methods have been developed for shot-gun ESI – tandem MS (MS/MS)  and LC-MS/MS analysis [21,22]. Gas chromatography (GC)-MS also provides an excellent method for oxysterol and sterol analysis , but is less favoured in lipidomics laboratories.
Lipidomics and oxysterol biochemistry
Acknowledgement This work was supported by the UK Biotechnology and Biological Sciences Research Council (BBSRC, grant numbers BB/I001735/1 and BB/N015932/1 to WJG, BB/L001942/1 to YW).