Isolation, purification and characterization of proteoglycans from deep sea shark, Echinorhinus brucus (Bramble shark) cartilage

Abstract

The present study was aimed at isolation of proteoglycans from deep sea shark cartilage, Echinorhinus brucus and characterization of their glycosaminoglycans and protein. PGs are important macromolecules of extracellular matrix involved in various cellular functions including cell growth and differentiation. PGs were isolated by chaeotropic digestion and anion-exchange chromatography with DEAEsephacel. Chemical composition such as protein and GAGs were determined by colorimetric assays. SDS-PAGE and chromatographic methods were implemented for further characterization. Trypsin digested samples were used to characterize the protein by peptide based LC-MS/MS analysis. Sample was injected into Q Exactive Plus Bench top Orbitrap instrument coupled to Thermo Easy nLC 1200. The acquired data was analysed in Proteome Discoverer 2.1SP1 on Sequest HT search engine and NCBI full protein database. GAG characterization was done with positive and negative ion full scanning of sample and standard with aid of MS/MS QTRAP 4000 model. NCBI protein data base revealed the presence of aggrecan core protein, decorin and epiphycan peptide hits in the proteoglycans sample and similar ion patterns were noticed for GAG isolated from sample compared to standard GAGs. In chemical composition, total protein, total carbohydrate, uronic acid, hexosamine and sulfated GAGs values were observed to be 0.468 mg/mg, 0.530 mg/mg, 0.098 mg/mg, 0.20 mg/mg and 0.06 mg/mg, respectively. According to the results, it was clear that in AGE both high molecular and low molecular weight PG were present in the isolated sample. Presence of GAGs was confirmed by PAGE using standard GAGs. These methods can be used to characterize the chemical and structural characterization of proteoglycans. In conclusion PGs isolated in our study were found to be pure as confirmed by structural and chemical characterization.