Mass spectrometry (MS) allows the detection and structural characterisation of intact molecules such as fatty acids and complex lipids. Desorption electrospray ionization (DESI) is an ambient ionization technique used for MS analysis and profiling and imaging of drugs, metabolites and lipids directly from biological samples with no sample preparation. With the recent introduction of morphologically friendly DESI-MS solvent systems, it is also possible to acquire DESI-MS data non-destructively. Due to the extractive nature of these solvent combinations, enough ion intensity can be generated to chemically profile samples of microscopic dimensions. The objective of this work was to perform chemical profiling on intact bovine blastocysts by DESI-MS, focusing on lipid distributions. Blastocysts produced in vitro were washed 3 times in PBS + 0.1% polyvinyl alcohol to remove lipids present in the culture medium, were placed in PBS/methanol 50% and stored under –20°C for 1 week. For DESI-MS analysis, the embryos were simply placed in glass slides and allowed to dry at room temperature. Mass spectra were acquired in the negative ion mode at the mass/charge range from m/z 150 to 1000, using as solvents a combination of 1:1 (vol/vol) ethanol:dimethylformamide (DMF) or acetonitrile:DMF. The mass spectrometer used was a LTQ linear ion trap mass spectrometer controlled by Xcalibur 2.0 software (Thermo Fisher Scientific, San Jose, CA, USA). The lipid species detected included deprotonated free fatty acids such as palmitic acid (m/z 255.2), stearic acid (m/z 283.2), arachidonic acid (m/z 311.2) and docosanoic acid (m/z 339.3). Free fatty acid dimers appear in the region from m/z 500 to 650 and complex lipids represented mainly by glycerophospholipid classes appear in the region from m/z 700 to 1000 and include phosphatidylinositols (PI 38:1; m/z 788.7), phosphatidylserines (PS 36:1, m/z 885.7) and also the chlorinated phosphatidylcholines (PC 36:1; m/z 794.7). After recording the mass spectra, embryos could still be observed in the glass slide with evident dehydration due to the action of the organic solvent. Since lipid composition of bovine embryos is closely related to cryosensitivity and due to the limited amount of analytes (each embryo is estimated to have a mass of 15 pg of total lipids) lipid analysis usually involves the pooling of individuals to have a large enough amount of analytes. Traditionally, gas chromatography is used for fatty acid residue analysis in oocytes and embryos pooled are submitted to lipid extraction and derivatization. Mass spectrometry by DESI, however, allows direct analysis of intact and single embryos and the profiling of not only free fatty acids but also complex lipids, represented mainly by 3 glycerophospholipid classes (PC, PI and PS). We envisage that DESI-MS will likely become a routine tool for the analysis of lipid composition in mammalian embryos and will contribute significantly to the development of culture systems that produce embryos with higher cryoresistance.
Support from the Purdue University Center for Cancer Research Small Grants Program is gratefully acknowledged.
Chemical Compounds from the Preanal Gland Secretions of the Male Tree Agama (Acanthocercus atricollis) (Fam. Agamidae)
