This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.In order to define the functional roles of glycan expression during disease formation it is necessary to determine their patterns of expression as a function of tissue location. The structural heterogeneity of glycosaminoglycans (GAGs) is of interest because it can be related to various biological parameters such as aging and osteoarthritis. The present work applies high resolution capillary amide-80-LC with on-line tandem MS to the analysis of cartilage tissue corresponding to one microgram GAG. A structural analysis of the abundant internal delta unsaturated oligosaccharides, lower abundance linker region, and saturated oligosaccharides in normal and osteoarthritic cartilage samples is described. Isotopic labeling and normal phase-LC tandem MS analysis of GAG oligosaccharides allows for structural characterization and quantification of different glycoforms. Sulfated GAGs are extracted from papain-digested juvenile bovine and adult human cartilage samples (3 replicates of 1 microg, 3 microg, and 5 microg GAG equivalent) using a streamlined multi-step procedure. Samples were released from residual core protein, passed through a C18 reversed phase solid phase extraction column, ethanol precipitated, enriched using a strong anion exchange column, and desalted using an ethanol precipitation to extract GAGs from the intact cartilage. Oligosaccharides were partially depolymerized followed by derivatization with 2-anthranilic-3,4,5,6-d4 acid. Derivatized cartilage derived GAG samples (cartilage-d4-2AA) were cleaned, spiked with standard CSA-d0-2AA, and subjected to an online capillary amide-80-LC/MS/MS platform. A combination of MS analysis and tandem MS analysis was employed for detailed structural characterization.
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