65 THREE-DIMENSIONAL CHEMICAL IMAGING OF A WHOLE PIG FETUS BY DESORPTION ELECTROSPRAY IONIZATION MASS SPECTROMETRY

Abstract

Desorption electrospray ionization (DESI) is an ambient ionization technique in mass spectrometry (MS) used for 2-dimensional (2D) imaging of drugs, metabolites and lipids directly from biological samples such as tissue sections with high chemical specificity and no need for sample preparation. From a selected representative set of 2D images it is possible to construct 3-dimensional models (3D) to illustrate the spatial distributions of specific biomolecules. Recently, the use of a dimethylformamide-based solvent system was introduced, which allows non-destructive DESI-MS analysis of tissue sections. This work describes the application of 3D DESI-MS chemical imaging capability to allow the unambiguous matching of chemical and morphological information in a whole swine fetus at the end of the embryonic phase of the gestational period. Fifty longitudinal sections (15-μm thick) spaced by an average of 160 μm from a swine fetus were used for the experiments. Tissue sections were stored under –80°C and placed in desiccator for 15 min before analysis. Mass spectra were directly acquired in the negative ion mode at the mass-to-charge (m/z) range of 150 to 1000 using 1:1 (vol/vol) acetonitrile/DMF as solvent at 1.0 μL min^–1 flow rate and 300 μm of spatial resolution. The mass spectrometer used was an LTQ linear ion trap controlled by Xcalibur 2.0 software (Thermo Fisher Scientific, San Jose, CA, USA). An in-house program allowed the conversion of mass spectra files into a format compatible with BioMap (freeware; Novartis International AG, Basel, Switzerland), used to generate 2D ion images of specific m/z values. The 2D images were processed and exported to the software 3D-Doctor (Able Software Corp., Lexington, MA, USA), on which the 3D models were built. Extensive chemical information was unambiguously matched with morphological features by hematoxylin and eosin (H&E) staining of samples after DESI-MS analysis. The lipid species detected in the mass spectra were deprotonated free fatty acids, fatty acid dimers and complex lipids represented mainly by glycerophospholipids, which were identified by tandem MS experiments. Multiple lipid species were chosen to construct 3D models, such as the ion m/z 788.7 (phophatidylserine 36:1) and the ion m/z 885.8 (phosphatidylinositol 38:4). Several structures of the swine fetus were chemically distinguished, such as the nervous system (brain and spinal cord), liver, heart and connective tissue, within others. Statistical multivariate analysis was additionally used to better individualize chemical features observed in the fetus body chemical imaging and to correlate the organs lipid profiles to their embryologic origin. Since DESI-MS 3D models allow the investigation of a variety of lipid species related to specific organs without the need of fluorescent or radioactive labelling, we expect that this approach will be used to increase the understanding and the development of strategies to avoid aberrations in organogenesis observed in fetuses generated by biotechnologies such as nuclear transfer and transgenesis.

Supported by ACS IRG Purdue University Center for Cancer Research.