Graphitized Carbon LC-MS Characterization of the Chondroitin Sulfate Oligosaccharides of Aggrecan Ruby P. Estrella, John M. Whitelock,* Nicolle H. Packer, † and Niclas G. Karlsson ‡ Proteome Systems Ltd., Locked Bag 2073, North Ryde Sydney, NSW 1670 Australia, and Graduate School of Biomedical Engineering, Level 5, Samuels Building, University of New South Wales, Sydney, NSW 2052 Australia A novel in-gel endoglycosidase technique to study oli- gosaccharides with graphitized carbon LC-MS has re- vealed differences in the sulfation profile between the linkage and repeat regions of chondroitin sulfate on aggrecan. Bovine articular cartilage aggrecan was isolated in a composite agarose PAGE gel or diluted in ammonium acetate buffer and was digested overnight with chondroiti- nase ABC. Including a chemical release/reduction pro- tocol after digestion, we could separate and detect three differentially sulfated chondroitin sulfate disaccharides of the repeat region (ΔUA1-3GalNAc0/4/6S-ol) from the three differentially sulfated linkage region hexasaccha- rides (ΔUA1-3GalNAc0/4/6S1-4GlcA1-3Gal1-3Gal1- 4Xylitol). Graphitized carbon LC-MS in the negative ion mode was able to resolve isomeric disaccharides and linkage region hexasaccharides. Specific MS 2 and MS 3 enabled us to confirm the sulfate location on all oligosac- charides by comparing their fragmentation with sulfated disaccharide standards. The presence of unsulfated, 6-sulfated, and 4-sulfated linkage regions was correlated with positive Western blot staining with the respective CS linkage region neoepitope antibodies (1B5, 3B3, 2B6) on digested aggrecan. Our strategy of examining linkage region and repeat region profiles is applicable to screening GAGs from various biological samples in order to detect differences between normal and disease states. Glycosaminoglycans (GAGs) are the covalently attached sul- fated oligosaccharide chains of proteoglycans (PGs) and confer many of the biological functions of PGs. Some of their important roles include cell signaling, tissue development, inflammation, and cartilage integrity. 1-4 In normal cartilage pathology, the most predominant PG, aggrecan, interacts with hyaluronic acid and collagen fibers to provide a stable supportive scaffold. 5 The negatively charged sulfate groups of chondroitin sulfate (CS) on aggrecan create a strong electrostatic repulsion leading to a hydrophilic environment that contributes to cartilage resistance to compression. However, in disease states such as osteoarthritis, there is a breakdown in proteoglycans causing a disruption in the balanced framework in cartilage, and one of the first hallmarks of disease is the release of proteoglycan protein and GAG fragments into the synovial fluid. 5,6 Therefore, understanding the structural complexity of CS and other GAGs is imperative to detecting these fragments in an early diagnostic situation. In addition to CS, the GAG family of oligosaccharides includes the following: dermatan sulfate (DS), heparan sulfate (HS), heparin (HP), and keratan sulfate (KS). Another type of GAG, hyaluronic acid (HA), is an exception as it is unsulfated and not attached to a protein. One of the major obstacles to studying the GAG components of proteoglycans derived from biological samples is their low abundance, heterogeneous size, molecular weight, and charge distribution. Analysis may also be complicated due to the fact that some PGs, such as aggrecan and perlecan, may be populated by more than one type of GAG. 7,8 Performing initial anion-exchange chromatography can eliminate some non-PG components and enrich for proteoglycans; however, further separation can be achieved with the use of 1D composite gel electrophoresis combined with oligosaccharide detection stains such as alcian blue or toluidine blue. 9 As the most prevalent GAG of aggrecan, CS occupies up to 100 sites on the protein core. 10 The main repeat region of CS is composed of alternating glucuronic acid (GlcA) and N-acetylga- lactosamine (GalNAc) subunits making up linear oligosaccharide chains up to 20 kDa each, and these are attached to serine residues on the protein core via a tetrasaccharide linkage region sequence GlcA1-3Gal1-3Gal1-4Xyl. There are up to 25 varia- tions of CS polymers due to modifications which include the following: unsulfation, 4- or 6- sulfation on the GalNAc, 2-sulfation on the GlcA, and epimerization of GlcA to iduronic acid (IdoA). 11 While there are inherent tissue- or species-specific differences in * To whom correspondence should be addressed. Tel: +612 93853948. Fax: +612 96632108. E-mail: j.whitelock@unsw.edu.au. † Current address: Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, Australia. ‡ Current address: Centre for Bioanalytical Science, Chemistry Department, NUI Galway, Ireland. (1) Hardingham, T. E.; Fosang, A. J. FASEB J. 1992, 6, 861-870. (2) Caterson, B.; Mahmoodian, F.; Sorrell, J. M.; Hardingham, T. E.; Bayliss, M. T.; Carney, S. L.; Ratcliffe, A.; Muir, H. J. Cell Sci. 1990, 97 (Pt 3), 411-417. (3) Taylor, K. R.; Gallo, R. L. FASEB J. 2006, 20,9-22. (4) Whitelock, J. M.; Iozzo, R. V. Chem. Rev. 2005, 105, 2745-2764. (5) Roughley, P. J. 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