AN IMPROVED PLATFORM FOR GAG DISACCHARIDE ANALYSIS USING LC/MS

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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. The structurally-polydisperse heparan sulfate (HS) chains are responsible for the interactions of HS proteoglycans with a wide variety of proteins, playing critical roles in biology. One HS biosynthesis step, the coordinated N-deacetylation/ N-sulfation on the GlcNAc residues, can be interrupted by the limited supply of 3'-phosphoadenosine 5'-phosphosulfate, and result in the formation of unsubstituted glucosamine residues within HS chains. Although endogenous N-unsubstituted saccharides have only been identified in a handful of studies, they have been implicated to be important in cellular and pathophysiological phenomena. Therefore, there is a clear need to systematically study the occurrence, and determine the structure and distribution patterns of these free-amino-containing HS disaccharides from mammals in a tissue-specific manner. At the present time, all samples received by the MSR for HS analysis are extracted and then digested exhaustively heparin lyases. The disaccharides are then profiled using SEC/MS. The data are used to determine the average chain lengths and average incorporation of sulfate and acetate groups. The data serve to identify the distribution of disaccharides in the internal regions of the parent chain as well as the non-reducing end. It is the non-reducing end that is of particular interest for protein binding studies. Update on progress: The SEC LC/MS system is used for disaccharide analysis of every sample received in the laboratory. It has been used for several core and collaborative studies that have been published (1-5). We have tested new Waters UPLC SEC stationary phases for use with GAG disaccharide analysis. The goal is to be able to scale down the column diameter using the UPLC SEC material, taking advantage of its increased efficiency. In initial studies, a UPLC SEC column appropriate for proteins was not appropriate for GAG disaccharides. We are waiting for Waters to release a smaller pore size UPLC SEC solid phase. It is necessary to increase the chromatographic resolution, sensitivity, and to reduce analysis time for GAG disaccharide analysis. These capabilities are needed in particular for analysis of GAGs from tissue surfaces and from tissue microarrays. Towards these ends, we are testing both UPLC HILIC and Chip-based porous graphitized carbon chromatography. 1. Staples, G. O., Shi, X., and Zaia, J. (2011) Glycomics Analysis of Mammalian Heparan Sulfates Modified by the Human Extracellular Sulfatase HSulf2, PLoS ONE 6, e16689. 2. Schumacher, V., Schlotzer-Schrehardt, U., Karumanchi, S. A., Shi, X., Zaia, J., Jeruschke, S., Zhang, D., Pavenstaedt, H., Drenckhan, A., Amann, K., Ng, C., Hartwig, S., Ng, K.-H., Ho, J., Kreidberg, J. A., Taglienti, M., Royer-Pokora, B., and Ai, X. (2011) WT1 regulation of Sulf expression is crucial to maintaining the glomerular filtration barrier, Journal of the American Society for Nephrology Accepted 2/22/11. 3. Staples, G. O., Shi, X., and Zaia, J. (2010) Extended NS domains reside at the non-reducing end of heparan sulfate chains, J Biol Chem 285, 18336-18343. 4. Langsdorf, A., Schumacher, V., Shi, X., Tran, T., Zaia, J., Jain, S., Taglienti, M., Kreidberg, J. A., Fine, A., and Ai, X. (2010) Expression regulation and function of Sulfs in the spermatogonial stem cell niche, Glycobiology 21, 152-161. 5. Shi, X., and Zaia, J. (2009) Organ-specific heparan sulfate structural phenotypes, J Biol Chem 284, 11806-11814.