MICRO-HPLC AND LC/MS METHODS FOR NEURAL SYSTEMS

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Neurological research depends ultimately on the accurate analysis of cellular components. The objective of this research program is to develop combined on line chromatographic-mass spectrometric procedures with the highest achievable sensitivity for glycoconjugates and lipids in order to contribute to the understanding of the nervous system. We postulate that developmentally regulated interactions between cell-surface glycoconjugates and complementary cell-surface or extracellular ligands participate in the regulation of specific morphogenetic events and that the membrane environment or lipid composition of cellular membranes influences these interactions. Biological systems, suitable for studies of neural differentiation, under investigation in this institution include: glycoconjugates and cell classes of the developing mouse neocortex, regulation of myelin phospholipid synthesis, developmental studies of peripheral nerve, and glycolipid metabolism in tissue culture cells of neural origin. The nature of these systems dictate that the limits of analytical technology be pursued in order to obtain quantitative chemical information on a scale sufficiently small to allow meaningful chemical-morphological correlations. Mass spectrometry can provide specific structural information and/or high sensitivity measurements. In pursuit of these chemical limits, the following approaches for glycolipid, phospholipid and glycoprotein high mannose oligosaccharides analysis will be investigated: (1) Establish conditions for the liquid chromatographic-mass spectrometric (LC/MS) analysis of underivatized phospholipid classes and molecular species, (2) Develop methods for the LC/MS analysis of underivatized and/or permethylated glycosphingolipids and also conditions for the analysis of the perbenzoylated derivatives, (3) Devise procedures for the LC/MS analysis of underivatized, permethylated and N-trifluoroacetyl-methylated high mannose oligosaccharides and (4) Investigate alternative methods of ionization for mass spectrometric analysis which are potentially useful for analysis of phospholipids, glycolipids and oligosaccharides.