A tissue extraction procedure for GAGs compatible with LC/MS from Rat Tissues
Glycosaminoglycans (GAGs) are long, unbranched sugar chains covalently bound to the core proteins of proteoglycans. They are abundant both on the cell surface and in the extracellular matrix. Heparan sulfate (HS) and chondroitin sulfate (CS) serve a wide range of biological functions, including cell-surface binding of growth factors and their sequestration in extracellular matrices. The structures of HS and CS exhibit high polydispersity in disaccharide composition, sulfation pattern, chain length, and epimerization pattern, due to their non-template driven biosynthesis..
This work describes comparative analysis of CS and HS among rat organ tissues using a novel amide-silica LC/MS glycomics platform. The extraction procedure combined with stable isotope labeling and LC/MD enables compariative GAG glycomics among mammalian organ tissues.
Dry tissues (10-50 mg) from liver, brain, muscle, heart, kidney, lung and spleen of rats were digested with pronase and benzonase. Both chondroitin sulfate and heparan sulfate were recovered by diethylaminoethyl (DEAE) anion exchange column. The targeting GAGs were recovered by another DEAE procedure after exhaustive enzyme digestion of other forms of GAGs. These GAGs are partially or completely digested, followed by stable isotopic tagging. These samples were chromatographically separated by an amide-based HILIC capillary column (250 micron i.d.) before spayed into an Esquire 3000 QIT or Q-TOF mass spectrometer. Both MS and MS2 were utilized to analyze disaccharides and oligosaccharides.
The primary challenge to MS-based glycomics is removal of tissue components that cause spectral background and degrade instrumental performance over time. In this study, a trapping column is used to concentrate GAG components and wash away contaminants. Tetraplex glycomics tags were used to label the saccharides. A reference GAG mixture was labeled with the d0 form of the tag and used as an internal standard for all organ tissue samples. The disaccharides profiles of both chondroitin sulfate and heparan sulfate and some of the domain information of these GAGs of seven tissues from rat were obtained.
These results demonstrate that statistically different GAG disaccharides distribution for tissues from different sources. This platform will be used to recover and analyze GAGs from other biological matrices, and examine samples that are related to disease states.
