This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Dermatan/chondroitin sulfate (DS/CS) proteoglycans are expresssed in many mammalian tissues and the DS forms are predominant in skin. DS is particularly interesting because of the biological processes it is implicated in, such as wound repair, infection, cardiovascular disease, tumorigenesis, and fibrosis. DS contains repeating units of iduronic acid (IdoA) and N-acetylgalactosamine (GalNAc), IdoA(1,3)GalNAc(1-4), and may be sulfated at the 4- and 6- positions of GalNAc and 2-position of IdoA. Epimerization at the 5th position of uronic acid ring in chondroitin sulfate (CSB) differentiates it from chondroitin sulfate A (CSA) and chondroitin sulfate C (CSC). Three parts compose linear polysaccharide GAG chain: the linker, consisting of a GlcA-Gal-Gal-Xyl- attached to serine residue of the core protein, followed by the main body, and the non-reducing end. This study concerns sequencing the main body and the non-reducing end of DS chains. Methods CSA (GlcA, GalNAc-4-sulfate), CSB (IdoA, GalNAc-4-sulfate), CSC (GlcA, GalNAc-6-sulfate), and all enzymes were obtained from Seikagaku America/Associates of Cape Cod (Falmouth, MA). Unsaturated oligosaccharides were generated by digestion of DS with chondroitinase ACI and/or ABC while saturated oligosaccharides were generated by digestion with testicular hyaluronidase. Oligosaccharides were partially purified using a Superdex peptide column (3.2 mm x 30 cm, Amersham Biosciences) and 0.1M ammonium acetate, 10% MeOH mobile phase. Negative mode mass spectra were acquired using an Applied Biosystems/MDS-Sciex API QSTAR Pulsar Qo-TOF mass spectrometer fitted with nanospray ion source. CID spectra were obtained at collision energy of -18V where the precursor ion remained abundant. Results and Discussion The variable placement of IdoA and sulfation positions on GalNAc residue poses a challenge in sequencing DS polysaccharide chains by MS. To determine the sulfate position and epimerization, we compared the CID ion abundances of the fragment ions formed from DS relative to those produced from standards with known epimerization and GalNAc sulfate positions. Previous studies indicated that the abundances of glycosidic bond cleavages formed from CID of DS/CS reflect GalNAc residues sulfation positions. Moreover, ions formed from ring cleavage of the unsaturated GlcA indicate epimerization. The tetramer series of several DS and decorin enabled us to look at only one uronic acid with its epimerization state. CID spectra of the unsaturated tetramer isolated after lyase digestion of CSA, CSC, and CSB, respectively, display ions suggesting sulfate position and epimerization. Ions B31- and Y11- reflect CSA-like content, 0,2X32- and Y32- ions display CSB-like epimerization state, and C32- and (M-SO3)2- ions indicate CSC-like sulfate position. The non-reducing tetramer is 18 u higher in mass than the main body tetramer by 18 u. The saturated CS standards of known sulfation position were obtained from hyaluronidase digestion. Characteristic ions B31- and Y11- indicate CSA-like sulfation pattern, C32- and (M-SO3)2- ions suggest CSC-like content while Y32- ions indicate CSB-like epimerization state. Ring cleavage ions, 0,2X32- , were not observed, suggesting that ?,?-unsaturation of GlcA is crucial in the formation of 0,2X32- ions. The sulfate position and epimerization of the hexamer and octamer series of DS and decorin are being determined to test the viability of this method.