Dermatan sulfate (DS) chains are variants of chondroitin sulfate (CS) that are expressed in mammalian extracellular matrices and are particularly prevalent in skin. DS has been implicated in varied biological processes including wound repair, infection, cardiovascular disease, tumorigenesis, and fibrosis. The biological activities of DS have been attributed to its high content of IdoA(alpha1-3)GalNAc4S(beta1-4) disaccharide units. Mature CS/DS chains consist of blocks with high and low GlcA/IdoA ratios, and sulfation may occur at the 4- and/or 6-position of GalNAc and 2-position of IdoA. Traditional methods for the analysis of CS/DS chains involve differential digestion with specific chondroitinases followed by steps of chromatographic isolation of the products and di-saccharide analysis on the individual fraction. This work reports the use of tandem mass spectrometry to determine the patterns of sulfation and epimerization of CS/DS oligosaccharides in a single step. The approach is first validated and then applied to a series of skin DS samples and to decorins from three different tissues. DS samples ranged from 74 to 99% of CSB-like repeats, using this approach. Decorin samples ranged from 30% CSB-like repeats for those samples from articular cartilage to 75% for those from sclera. These values agree with known levels of glucuronyl C5-epimerase in these tissues.

Carbohydrates of all classes consist of glycoform mixtures built on common core units. Determination of compositions and structures of such mixtures relies heavily on tandem mass spectrometric data. Analysis of native glycans is often necessary for samples available in very low quantities and for sulfated glycan classes. Negative tandem mass spectrometry (MS) provides useful product ion profiles for neutral oligosaccharides and is preferred for acidic classes. In previous work from this laboratory, site-specific influences of sialylation on product ion profiles in the negative mode were elucidated. The present results show how the interplay of two other acidic groups, uronic acids and sulfates, determines product ion patterns for chondroitin sulfate oligosaccharides. Unsulfated chondroitin oligosaccharides dissociate to form C-type ions almost exclusively. Chondroitin sulfate oligosaccharides produce abundant B-and Y-type ions from glycosidic bond cleavage with C-and Z-types in low abundances. These observations are explained in terms of competing proton transfer reactions that occur during the collisional heating process. Mechanisms for product ion formation are proposed based on tandem mass spectra and the abundances of product ions as a function of collision energy. (J Am Soc Mass Spectrom 2007, 18, 952-960) © 2007 American Society for Mass Spectrometry T he development of tandem mass spectrometric methods for glycomics requires clear understanding of glycan fragmentation mechanisms. Released glycans are heterogeneous with regard to presence of acidic residues or modifying groups. Such acidic moieties strongly influence the energetics of fragmentation and the patterns of tandem mass spectrometric product ion formation. Glycosaminoglycans (GAGs) consist of repeating disaccharide units, several classes of which are sulfated. Oligosaccharides of composition (Gal6X␤4GlcNAc6S␤3) n where X ϭ H or SO 3 H and S ϭ SO 3 H (derived from keratan sulfate), dissociate for form abundant A-type cross-ring cleavages to the reducing terminal residue [1,2]. Oligosaccharides of composition ⌬(HexA␤3GalNAc4/6S) n (derived from chondroitin sulfate, CS) produce B-and Y-type ions [3][4][5][6][7], the pattern of which depends on the charge state [8]. Those of composition ⌬(HexA2X␣/␤4GlcNY6Z) n where X, Z ϭ H or SO 3 H and Y ϭ Ac, H, or SO 3 H (derived from heparan sulfate) produce a complex mixture of A-, B-, C-, X-, Y-, and Z-type product ions [9 -12]. The goal of this work is to describe mechanisms behind the formation of negative product ions for GAG oligosaccharides.Nonsulfated native glycans dissociate in the positive mode to form abundant ions via glycosidic cleavages of the B-and Y-types, resulting from association of a cation with the glycosidic oxygen atom [13][14][15]. The most facile cross-ring cleavages are those to 4-or 6-linked reducing terminal residues [16 -18]. Although cross-ring cleavage ions are abundant for 4-linked reducing terminal residues, those within internal branching residues have significantly lower abund...

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