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. Introduction: Glycomics, known as the study of the structure and function of glycans, is a rapidly growing field. Glycosaminoglycan (GAG) chains are made of repeating disaccharide units that are attached to proteoglycan core proteins on adherent animal cell surfaces and in extracellular matrices. Chondroitin sulfate (CS) is a glycosaminoglycan that consists of repeating disaccharide units of [(GlcA?(1-3)GalNAc?(1-4)]. Three types of CS exist, CS-A, CS-B, and CS-C. Presently, the field of glycomics lacks an effective analytical method for the isomeric differentiation and relative quantification of GAGs in small (1-10 microgram) biological samples. This work describes the development of a method for quantification of glycoforms using a stable isotopic labeling technique, and its application to the sulfated GAGs. Methods: CS samples, both standard and unknown, were partially depolymerized by chondroitin lyase ABC. The standard CS was then derivatized via a reductive amination reaction with 2-anthranilic acid, while the unknown CS was derivatized with 2-anthranilic-3,4,5,6-d4 acid. The derivatized CS samples were cleaned and the excess reagent removed via a cellulose microspin column. Equimolar mixtures of the standard and unknown CS samples were made. The isotopically labeled CS mixture was subjected to size exclusion liquid chromatography in a 10% acetonitrile, 50 mM ammonium formate buffer with on-line electrospray ionization mass spectrometrometric detection in the negative mode. Automated tandem mass spectrometry was acquired and quantification of unknown samples was found using relative ion abundances of the diagnostic ions. Abstract: A sample of lyase digested CSA was used as a reference against which all unknown CS samples were compared. The reference sample was reductively aminated with d0-anthranilic acid and the unknown CS samples with d4-anthranilic acid. The samples were mixed and separated using SEC with on-line negative ESI MS/MS detection. The HPLC flow was split prior to the sample inlet, allowing 10 microliters/minute of flow into the mass spectrometer. Tandem MS was performed using the automated MSn feature of the ion trap. The isolation and fragmentation windows were set to 12.0 u so that CID spectra of both heavy and light forms were acquired simultaneously. Tandem MS resulted in Y ions and [M-H-SO3] ions containing the reducing end and differing by four mass units, and B ions that are isobaric for both heavy and light forms of AA-labeled CS oligosaccharides. The abundances of Y and [M-H-SO3] heavy and light ions were used for glycoform distribution predictors for unknown CS samples. The method is validated by acquiring automated tandem mass spectra on several isotopically labeled CS mixtures in triplicate. The mixtures were analyzed and the percent total ion abundances of light and heavy predictive ions containing the reducing end were calculated. Light and heavy predictive ion contributions from the unknown were then put into a set of three equations. The three equations were solved for three unknowns that represent the percentage of CSA, CSB, and CSC in a mixture. The results demonstrate that tandem mass spectrometry can be used for the isotopic quantification of glycoforms of chondroitin sulfate.
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