Analysis of novel over- and under-sulfated glycosaminoglycan sequences by enzyme cleavage and multiple stage MS

• Published in: Proteomics (Weinheim) 2009-07, Vol.9 (13), p.3435-3444
• Main Authors: Zamfir, Alina D, Flangea, Corina, Sisu, Eugen, Serb, Alina F, Dinca, Nicolae, Bruckner, Peter, Seidler, Daniela G
• Format: Article
• Language: English
• Subjects: Analytical, structural and metabolic biochemistry; Biological and medical sciences; Carbohydrate Sequence; Chondroitin Lyases - metabolism; Decorin; Extracellular Matrix Proteins - isolation & purification; Fundamental and applied biological sciences. Psychology; Glycosaminoglycans; Glycosaminoglycans - analysis; Glycosaminoglycans - chemistry; Glycosaminoglycans - metabolism; Humans; Miscellaneous; Multistage mass spectrometry; Oligosaccharides - analysis; Oligosaccharides - chemistry; Oligosaccharides - metabolism; Proteins; Proteoglycans - isolation & purification; Skin fibroblasts; Spectrometry, Mass, Electrospray Ionization - methods; Sulfation; Sulfur - chemistry; Tandem Mass Spectrometry - methods
• ISSN: 1615-9853; 1615-9861; 1615-9861
• DOI: 10.1002/pmic.200800440

We report on a novel strategy for identification of specific sulfation motifs in chondroitin/dermatan sulfate (CS/DS) chain derived from decorin (Dcn), based on enzyme cleavage and multistage MS (MSn). Released CS/DS chains were digested with chondroitin B and in parallel with AC I lyases to obtain oligosaccharides of known hexuronic acid (HexA) epimerization. The depolymerized chains were separated by gel filtration, and collected di- and hexasaccharides were analyzed by ESI MSn. MS² on bisulfated 4,5-Δ-HexAGalNAc revealed an additional sulfate ester group at 4,5-Δ-HexA. MS² data provided evidence upon GlcA sulfation in Dcn due to the fact that 4,5-Δ-HexA derived from GlcA after chondroitin AC I lyase treatment. Hexasaccharide screening in the MS¹ mode indicated direct correlation between the sulfate distribution and HexA epimerization. MSn performed on ions that, according to mass calculation, correspond to pentasulfated [4,5-Δ-HexAGalNAc(GlcAGalNAc)₂], trisulfated [4,5-Δ-HexAGalNAc(GlcAGalNAc)₂] with IdoA-derived 4,5-Δ-HexA at the nonreducing end, tetrasulfated [4,5-Δ-HexAGalNAc(IdoAGalNAc)₂] and monosulfated [4,5-Δ-HexAGalNAc(IdoAGalNAc)₂] with GlcA-derived 4,5-Δ-HexA at the nonreducing end rendered fragmentation patterns confirming the presence of over-, regular, and under-sulfated regions as well as structural motifs having both types of HexA sulfated within Dcn CS/DS.