Chondroitin sulfate (CS) is a glycosaminoglycan consisting of repeating uronic acid, Nacetylgalactosamine disaccharide units [(HexAβ/α(1-3) GalNAcβ (1-4)] n. CS chains are polydisperse with respect to chain length, sulfate content and glucuronic acid epimerization content, resulting in a distribution of glycoforms for a chain bound to any given serine residue. Usually, CS glycoforms exist, differing in sulfation position and uronic acid epimerization. This work introduces a novel LC/MS/MS platform for the quantification of mixtures of CS oligosaccharides. The CS polysaccharides were partially depolymerized and labeled with either the light (d 0 ) or heavy (d 4 ) form of 2-anthranilic acid (2-AA). Excess reagent was removed and mixtures of the CS standard (d 0 ) and unknown (d 4 ) were made. The CS mixture was subjected to size exclusion chromatography (SEC) with on-line electrospray ionization mass spectrometrometric detection in the negative ion mode. Tandem mass spectra were acquired and quantification of unknown samples within the mixture was made using relative ion abundances of specific diagnostic ions. High accuracy and precision of the glycomics platform were demonstrated using glycoform mixtures made from standard CS preparations. The CS glycoform analysis method was then applied to cartilage extract, versican, and several dermatan sulfate preparations. This work presents the first application of a glycomics platform for the quantification of CS oligosaccharide mixtures to obtain specific information on the positions of GalNAc sulfation and uronic acid epimerization.Glycomics is applied biology and chemistry that focuses on the structure and function of carbohydrates. Carbohydrates have vital functions in the body; in particular, glycosylation serves to diversify protein functions. Despite their abundance, much less information is known about carbohydrates, as compared to genes and proteins, largely due to the absence of a simple code that determines their structures. Mass spectrometry (MS), high performance liquid chromatography and other analytical tools have advanced rapidly to support the growth of biomolecular applications in the field of proteomics. However, technologies and methods to address the most challenging problems in glycomics, particularly with respect to glycoform quantitative measurements, remain undeveloped.Glycosaminoglycans (GAGs) are linear polysaccharides that consist of repeating disaccharide units that are attached to proteoglycan core proteins on adherent animal cell surfaces and in extracellular matrices (1-5). GAGs play significant roles in the modulation of cellular signals through interactions with proteins, including growth factors and growth factor receptors (6-8). They mediate cell-cell and cell-matrix interactions and are crucial to cell development and

A key challenge to investigations into the functional roles of glycosaminoglycans (GAGs) in biological systems is the difficulty in achieving sensitive, stable and reproducible mass spectrometric analysis. GAGs are linear carbohydrates with domains that vary in the extent of sulfation, acetylation and uronic acid epimerization. It is of particular importance to determine spatial and temporal variations of GAG domain structures in biological tissues. In order to analyze GAGs from tissue, it is useful to couple mass spectrometry with an on-line separation system. The purposes of the separation system are both to remove components that inhibit GAG ionization and to enable the analysis of very complex mixtures. This contribution presents amide-silica hydrophilic interaction chromatography (HILIC) in a chip-based format for LC/MS of heparin, heparan sulfate and chondroitin/dermatan sulfate GAGs. The chip interface yields robust performance in the negative ion mode that is essential for GAGs and other acidic glycan classes while the built-in trapping cartridge reduces background from the biological tissue matrix. The HILIC chromatographic separation is based on a combination of the glycan chain lengths and the numbers of hydrophobic acetate groups and acidic sulfate groups. In summary, chip based amide-HILIC LC/MS is an enabling technology for GAG glycomics profiling.

Homeostasis of connective joint tissues depends on the maintenance of an extracellular matrix, consisting of an integrated assembly of collagens, glycoproteins, proteoglycans and glycosaminoglycans (GAGs). Isomeric chondroitin sulfate (CS) glycoforms differing in position and degree of sulfation and uronic acid epimerization play specific and distinct functional roles during development and disease onset. This work profiles the CS epitopes expressed by different joint tissues as a function of age and osteoarthritis. Glycosaminoglycans were extracted from joint tissues (cartilage, tendon, ligment, muscle and synovium) and partially depolymerized using chondroitinase enzymes. The oligosaccharide products were differentially stable isotope labeled by reductive amination using 2-anthranilic acid-d 0 or -d 4 and subjected to amide-HILIC on-line liquid chromatography-tandem mass spectrometry. The analysis presented herein enables simultaneous profiling of the expression of non-reducing end, linker region, and Δ-unsaturated interior oligosaccharide domains of the CS chains among the different joint tissues. The results provide important new information on the changes to the expression of CS GAG chains during disease and development.

Articular cartilage is a highly specialized smooth connective tissue whose proper functioning depends on the maintenance of an extracellular matrix consisting of an integrated assembly of collagens, glycoproteins, proteoglycans (PG), and glycosaminoglycans. Isomeric chondroitin sulfate glycoforms differing in position and degree of sulfation and uronic acid epimerization play specific and distinct functional roles during development and disease onset. This work introduces a novel glycosaminoglycan extraction method for the quantification of mixtures of chondroitin sulfate oligosaccharides from intact cartilage tissue for mass spectral analysis. Glycosaminoglycans were extracted from intact cartilage samples using a combination of ethanol precipitation and enzymatic release followed by reversed-phase and strong anion exchange solid-phase extraction steps. Extracted chondroitin sulfate glycosaminoglycans were partially depolymerized using chondroitinases, labeled with 2-anthranilic acid-d(4) (2-AA) and subjected to size exclusion chromatography with online electrospray ionization mass spectrometric detection in the negative ion mode. The method presented herein enabled simultaneous determination of sulfate position and uronic acid epimerization in juvenile bovine and adult human cartilage samples. The method was applied to a series of 13 adult human cartilage explants. Standard deviation of the mean for the measurements was 1.6 on average. Coefficients of variation were approximately 4% for all compositions of 40% or greater. These results show that the new method has sufficient accuracy to allow determination of topographical distribution of glycoforms in connective tissue.

This work describes improved workup and instrumental conditions to enable robust, sensitive glycosaminoglycan disaccharide analysis from complex biological samples. In the process of applying capillary electrophoresis with laser-induced fluorescence to glycosaminoglycan (GAG) disaccharide analysis in biological samples, we have made improvements to existing methods. These include (1) optimization of reductive amination conditions, (2) improvement in sensitivity through the use of a cellulose cleanup procedure for the derivatization and, (3) optimization of separation conditions for robustness and reproducibility. The improved method enables analysis of disaccharide quantities as low as 1 pmol prior to derivatization. Biological GAG samples were exhaustively digested using lyase enzymes, the disaccharide products and standards were derivatized with the fluorophore 2-aminoacridone and subjected to reversed polarity CE-LIF detection. These conditions resolved all known chondroitin sulfate disaccharides or eleven of twelve standard heparin/HS disaccharides, using 50 mM phosphate buffer, pH 3.5, and reversed polarity at 30 kV with 0.3 psi pressure. Relative standard deviation in migration times of CS ranged from 0.1% to 2.0% over 60 days, and the relative standard deviations of peak areas were less than 3.2%, suggesting that the method is reproducible and precise. The CS disaccharide compositions are similar to those obtained by our group using tandem mass spectrometry. The reversed polarity CE-LIF disaccharide analysis protocol yields baseline resolution and quantification of heparin/HS and CS/DS disaccharides from both standard preparations and biologically relevant proteoglycan samples. The improved CE-LIF method enables disaccharide quantification of biologically relevant proteoglycans from small samples of intact tissue.