Electrospray Ionization Tandem Mass Spectrometry for Direct Identification of Corneal Keratan Sulfate Oligosaccharides Without Prior Purification

Abstract

Abstract: : Purpose: In cornea, keratan sulfate (KS) is the major glycosaminoglycan (GAG), consisting of repeating disaccharide units composed of alternating residues of D–galactose (Gal) and N–acetyl–D–glucosamine (GlcNAc) linked ß–(1–4) and ß–(1–3), respectively. Abundant KS in the stroma is associated with corneal transparency. In this study, electrospray ionization tandem mass spectrometry (ESI–MS/MS) was employed to identify corneal keratan sulfate oligosaccharides. Methods: Non–sulfated, mono–, di–, tri–, and tetra–sulfated oligosaccharides derived from purified KS, sialic acid, and a mixture of shark cartilage KS oligosaccharides were infused into ESI–MS/MS. Bovine cornea KS was digested with keratanase II or endo–ß–galactosidase, and each digest was analyzed directly by ESI–MS/MS. Details of sources of standards, MS operating conditions, and programs for data analysis are described in Zhang, Y. et al. (2004), Analytical Chemistry (in press). Results: Two non–sulfated disaccharide isomers and two mono–sulfated disaccharide isomers were distinguished through MS/MS. In MS¹ spectra of multiply sulfated KS oligosaccharides, the charge state of the most abundant molecular ion equals the number of sulfates. Subsequent MS² and MS³ spectra of mono–, di–, tri–, and tetra–sulfated KS oligosaccharides and sialylated tetrasaccharides reveal diagnostic ions that can be used as structural fingerprints to identify the compositions of unknown KS oligosaccharides in a mixture from shark cartilage KS, and in enzyme digests of bovine corneal KS. Conclusions: This work presents the first evidence that ESI–MS/MS can be used to distinguish between KS disaccharide isomers, characterize KS oligosaccharide sequences, and identify the components of KS oligosaccharide mixtures without prior purification or chromatographic separation. In the accompanying paper by Schmack, I. Et al (ARVO # ), this technique is demonstrated to be applicable to identifying and quantifying disaccharides released from KS and CS/DS present in small portions of single sections of normal and LASIK–treated human corneas.

Keywords: cornea: stroma and keratocytes • cornea: stroma and keratocytes • cornea: stroma and keratocytes