Brain Chondroitin/Dermatan Sulfate, from Cerebral Tissue to Fine Structure: Extraction, Preparation, and Fully Automated Chip-Electrospray Mass Spectrometric Analysis
Chondroitin sulfate (CS) and dermatan sulfate (DS) glycosaminoglycans (GAGs) are covalently linked to proteins, building up a wide range of proteoglycans, with a prevalent expression in the extracellular matrix (ECM). In mammalian tissues, these GAG species are often found as hybrid CS/DS chains. Their structural diversity during chain elongation is produced by variability of sulfation in the repeating disaccharide units. In central nervous system, a large proportion of the ECM is composed of proteoglycans; therefore, CS/DS play a significant role in the functional diversity of neurons, brain development, and some brain diseases. A requirement for collecting consistent data on brain proteoglycan glycosylation is the development of adequate protocols for CS/DS extraction and detailed compositional and structure analysis. This chapter will present a strategy, which combines biochemical tools for brain CS/DS extraction, purification, and fractionation, with a modern analytical platform based on chip-nanoelectrospray multistage mass spectrometry (MS) able to provide information on the essential structural elements such as epimerization, chain length, sulfate content, and sulfation sites.
Key wordsGlycosaminoglycan Chondroitin sulfate Dermatan sulfate Cerebral tissue Chip-nanoelectrospray Multistage mass spectrometry
The authors are grateful to the Romanian National Authority for Scientific Research (Grants No. PN-II-RU-TE-2011-2-0008 and PN- II-ID-PCE-2011-3-0047) and German Society for Research (DFG SE1431/1-1 and IRTG1549/1 Molecular and Cellular Glycosciences) for financial support.
- 2.Sisu, E., Flangea, C., Serb, A., and Zamfir, A. D. (2011) Modern developments in mass spectrometry of chondroitin and dermatan sulfate glycosaminoglycans. Amino Acids. 41, 235–256.Google Scholar
- 10.Purushothaman, A., Fukuda, J., Mizumoto, S., ten Dam, G. B., van Kuppevelt, T. H., Kitagawa, et al. (2007) Functions of chondroitin sulfate/dermatan sulfate chains in brain development. Critical roles of E and iE disaccharide units recognized by a single chain antibody GD3G7. J. Biol. Chem. 282, 19442–19452.Google Scholar
- 14.Zamfir, A. D., Flangea, C., Altmann, F., and Rizzi, A. M. (2010) Glycosylation analysis of proteins, proteoglycans and glycolipids by CE-MS. Adv. Chromatogr. 49, 135–194.Google Scholar
- 15.Zamfir, A. D., Seidler, D., Kresse, H., and Peter-Katalinic, J. (2002) Structural characterization of chondroitin/dermatan sulfate oligosaccharides from bovine aorta by capillary electrophoresis and electrospray ionization quadrupole time-of-flight tandem mass spectrometry. Rapid Commun. Mass Spectrom. 16, 2015–2024.PubMedCrossRefGoogle Scholar
- 16.Flangea, C., Serb, A. F., Schiopu, C., Tudor, S., Sisu, E., Seidler, D. G., and Zamfir, A. D. (2009) Discrimination of GalNAc (4S/6S) sulfation sites in chondroitin sulfate disaccharides by chip-based nanoelectrospray multistage mass spectrometry. Cent. Eur. J. Chem. 7, 752–759.CrossRefGoogle Scholar