70 INCORPORATING MULTIPLE STAGES OF MASS SPECTROMETRY INTO LIPID PROFILING OF OOCYTES AND PRE-IMPLANTATION EMBRYOS

Abstract

Lipid profiling by mass spectrometry (MS) is increasingly used for the analysis of oocytes, embryos, and other reproductive cells. This analytical approach has several advantages, such as simple preparation (no need to perform extraction or separation), low detection limits (no need of sample pooling), and detection of structurally intact and diverse lipids. Many degrees of freedom are ensured by MS techniques (e.g. with the adoption of diverse ionization sources, mass analyzers, data acquisition systems), and this broadens the classes of lipids that can be detected and identified. Tandem or high-resolution MS experiments are normally performed for chemical characterisation. However, the use of novel approaches is a constant need to obtain deeper structural insights into lipids of biological interest, resulting in an information-rich dataset. Here we propose the use of multiple stages of MS for lipid profiling, specifically MS/MS data domain (i.e. ion mapping) experiments, so that comprehensive structural and relationship information (i.e. classes) can be extracted from a dataset. Indeed, the data generated have 2 dimensions of mass (i.e. precursor and product ions) and one of ion intensity, resulting in a datacube structure. Cutting through the datacube in different ways allows the extrapolation of (i) chemical composition of specific compounds (i.e. product scans) and (ii) pattern recognition for compounds that share identical neutral or charged fragments loss (i.e. neutral loss and precursor scans, respectively). The global chemical information enclosed into the datacube can be also processed by means of multiway statistical analyses to chemically characterise cells and cellular compartments. Preliminary data have been acquired, and the development of statistical tools for data processing is ongoing. Bovine and rat embryos were used for the experiments and analysed by extraction spray ambient MS. Experiments were performed with a Thermo Finnigan LTQ linear ion trap. Dimethylformamide-acetonitrile (1 : 1 v/v) was used as spray solvent. The ion mapping experiment was configured to scan ions of mass-to-charge (m/z) ratio 700 to 900 and perform MS/MS every m/z 1.5 window with a collision energy of 25 (arbitrary units). Fragments were detected in the m/z range of 150 to 900. Chemical differences are present between bovine and rat embryos, of note are palmitic, oleic, and stearic acids. The application of ion mapping to characterise species-specific and developmental dynamics regarding lipid composition is currently under investigation.