This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. C. elegans is being developed as a comparative glycomics platforms for the analysis N-glycans (NLO) and O-glycans (OLO), glycolipid-released glycans (GLO) and glycolipids. This is the first higher organism for which there is a complete description of its genome, anatomy and development. The structures of its major NLOs, OLOs, GLOs have been documented and there are mutants available with genetic deficiencies in each major pathway where glycan structural differences and other associated phenotypes have been observed. Many of these differences have been demonstrated in our hands using conventional glycomics methods. We are working to improve on conventional methods by the development of quantitative comparative glycomics platforms for the analysis of free glycans and glycopeptides. We are carrying out glycomics and lipidomics analyses of C. elegans for which there is considerable interest as a model in the study of metabolic diseases of glycosylation, ageing, development and host-pathogen interaction, all processes where glycosylation is important. We have characterized many of the N- and O-linked glycans and have found novel structures in each. In the current phase of the studies, we are concentrating on the glycolipids. Lysoglycerophosphocholine (Lyso-GPC) lipids are among the radyl-glycerophospho-lipids that are components of the cellular lipid bilayer in animal cells. Lyso-GPC lipids consist of a glycerophospho- choline backbone with one free glycerol hydroxyl at either sn-1 or sn-2 and a long-chain fatty acyl substituent. Lyso-GPC lipids were analyzed using a C18 column and the mobile phase A (H2O with 200 mM NH4HCO2) and B (CH3OH/(CH3)2CHOH, 2/3, v/v) by a NanoAcquity UPLC (Waters) coupled to a QStar Pulsar i Q-o-TOF MS (Applied Biosciences) by a TriVersa Nanomate (Advion). In positive ion mode, [M+H]+ ions for lyso-GPC lipids in lipid extracts from C. elegans were examined using high mass accuracy, high resolution measurements. Different subclasses of lyso-GPC lipids were detected in lipid extracts from C. elegans, for example, (1) 14:0a, 16:0a, 17:0a, 17:1a, 18:0a, 18:1a, 18:2a, 19:1a, and 20:1a/ lysoGPC, (2) Lyso/20:3a-, lyso/20:4a-, lyso/20:5a-, and lyso/21:4a-GPC, (3) 16:0e and 18:0e/lyso-GPC, but no p type of lyso-GPC. All lyso-GPC lipids from C. elegans eluted within the window of 15-20 min, with their order being based on fatty acyl chain length(s) and degree of unsaturation. CID of the [M+H]+ ions of all lyso-GPC lipids yielded an abundant product ion at m/z 184.06 that corresponds to the mass of the phosphocholine moiety present at the sn-3 position. This ion is a significant fragment that is characteristic for the polar head group of phosphocholine-containing lipids;this property suggests that it should be an important criterion for identification of glycerophosphocholine molecular species in biological lipid extracts. This method thus facilitates analysis of lyso-GPC lipids and can be used to explore their roles in C. elegans.
Showing the most recent 10 out of 253 publications
