Glycoform quantification of chondroitin/dermatan sulfate using a liquid chromatography-tandem mass spectrometry platform

Chondroitin sulfate (CS) is a glycosaminoglycan consisting of repeating uronic acid, N-acetylgalactosamine disaccharide units {[HexA beta/alpha(1-3)GalNAc beta(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 (do) and unknown (d4) were made. The CS mixture was subjected to size exclusion chromatography (SEC) with on-line electrospray ionization mass spectrometric 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. The 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 for obtaining specific information about the positions of GalNAc sulfation and uronic acid epimerization.