Glycosphingolipids (GSLs: 1-O-glycosides of the N-acyl-sphingosines, or ceramides) are a structurally and functionally diverse class of biological compounds distributed among all eukaryotes and some bacteria. Along with their biosynthetic intermediates, metabolic products, and simple non-glycosylated sphingosine derivatives, they are believed to be essential components of all eukaryotic cell membranes. A wide diversity in headgroup structures exists, contributing to the wide range of physical/chemical properties observed for GSLs. This, along with the complex sample matrix generally encountered in membrane lipid extracts, can render the quantitative extraction, accurate profiling and/or complete structure elucidation of all GSL components present in a tissue or organism daunting tasks. """"""""Universal"""""""" analytical strategies for GSL analysis have been proposed, but these are often highly labor intensive or address only a limited subset of GSL components or tissue types. Herein, development of a novel methodology for GSL analysis is proposed, incorporating goals of wide applicability, quantitative extraction, accurate representation of all components independent of headgroup properties, minimal loss of sensitive headgroup functional modifications, and compatibility with a variety of subsequent analytical instrumental and derivatization strategies. Further goals are ease of use and minimal sample handling for high-throughput applications, increased ionization of molecular species in mass spectrometric profiling, and adaptation to isotope-coding strategies enabling sensitive, reliable quantitative comparisons of GSL expression between tissues or cell types obtained, e.g., from different strains or developmental stages, or grown under different conditions. The proposed methodology is based on enzymatic removal of the ceramide N-acyl chain, followed by replacement with a suitable amino-reactive affinity tag. Biotin-containing tags will be used, enabling quantitative removal and subsequent release of all GSL components via immobilized streptavidin affinity systems. Initial development will be based on commercially available biotinylating reagents, but synthesis of reagents more specifically tailored for GSL applications, incorporating functional groups improving solubility, and promoting higher ionization yields, is proposed. In a later phase, synthesis of isotope coded affinity tags (sphingolipid ICAT, or SL-ICAT) is proposed, in order to facilitate quantitative glycosphingolipidomic profiling, analogous to the """"""""ICAT"""""""" methodology already developed for proteomic analysis. ? ?

Agency
National Institute of Health (NIH)
Institute
National Center for Research Resources (NCRR)
Type
Exploratory/Developmental Grants (R21)
Project #
5R21RR020355-02
Application #
6951395
Study Section
Special Emphasis Panel (ZRG1-BECM (01))
Program Officer
Sheeley, Douglas
Project Start
2004-09-27
Project End
2007-08-31
Budget Start
2005-09-01
Budget End
2007-08-31
Support Year
2
Fiscal Year
2005
Total Cost
$214,731
Indirect Cost
Name
University of New Hampshire
Department
Chemistry
Type
Schools of Engineering
DUNS #
111089470
City
Durham
State
NH
Country
United States
Zip Code
03824
Arigi, Emma; Blixt, Ola; Buschard, Karsten et al. (2012) Design of a covalently bonded glycosphingolipid microarray. Glycoconj J 29:1-12
Li, Yunsen; Arigi, Emma; Eichert, Heather et al. (2010) Mass spectrometry of fluorocarbon-labeled glycosphingolipids. J Mass Spectrom 45:504-19
Li, Yunsen; Thapa, Prakash; Hawke, David et al. (2009) Immunologic glycosphingolipidomics and NKT cell development in mouse thymus. J Proteome Res 8:2740-51
Li, Yunsen; Teneberg, Susann; Thapa, Prakash et al. (2008) Sensitive detection of isoglobo and globo series tetraglycosylceramides in human thymus by ion trap mass spectrometry. Glycobiology 18:158-65
Li, Yunsen; Zhou, Dapeng; Xia, Chengfeng et al. (2008) Sensitive quantitation of isoglobotriaosylceramide in the presence of isobaric components using electrospray ionization-ion trap mass spectrometry. Glycobiology 18:166-76
Castle, Sherry A; Owuor, Elizabeth A; Thompson, Stephanie H et al. (2008) Beta1,2-xylosyltransferase Cxt1p is solely responsible for xylose incorporation into Cryptococcus neoformans glycosphingolipids. Eukaryot Cell 7:1611-5
Klutts, J Stacey; Levery, Steven B; Doering, Tamara L (2007) A beta-1,2-xylosyltransferase from Cryptococcus neoformans defines a new family of glycosyltransferases. J Biol Chem 282:17890-9
Levery, Steven B (2005) Glycosphingolipid structural analysis and glycosphingolipidomics. Methods Enzymol 405:300-69