A number of analytical methods are being used in the Sanderson group to examine lipids and membranes. These include spectroscopy (Raman, SAXS), microscopy (confocal, Brewster angle, AFM) and mass spectrometry. In all cases, the aim is to reveal information on chemical processes (e.g. peptide/protein binding) or the chemical constitution of the sample.
In collaboration with Dr J A Mosely, we are developing analytical methods for the complete analysis of lipids. Complete analysis of glycerophospholipids requires identification of the headgroup and both acyl chains, including the number, position and geometry of double bonds. In early experiments we compared different methods for analysing lipids by MALDI-MSMS, including the effects of the matrix and additives to the matrix mixture. From this, specific pairs of related ions that could be used to distinguish the position of acyl chains on the glycerol backbone were identified, although it became clear that careful calibration is required for all experiments of this kind, as the intensities of key fragments are instrument-specific.1 More recently, we have developed analytical methods for the analysis of peptide-lipid samples, using liquid chromatography combined with tandem MS. This work has been fundamental in identifying a new process that occurs through transfer of acyl groups from lipids to peptides.2,3
Linear dichroism (LD) spectroscopy, using liposomal membranes aligned by shear flow, has proved to be a useful tool for monitoring the kinetics of peptide binding to membranes.4 For the analysis of lipid membrane composition, Raman spectroscopy of single liposomes held in optical tweezers was demonstrated by us to be a useful analytical tool (in collaboration with Dr A D Ward).5
We have combined thin-film methodologies such as Brewster angle microscopy (BAM, with Prof A Beeby) and AFM of protein films deposited onto ODS-treated silicon. This has enabled us to examine the membrane properties of viral matrix proteins.6 In collaboration with Dr R Kataky, scanning electrochemical microscopy (SECM) has been deployed to study the channel gating properties of a gramicidin analogue modified with nicotinamide.7