In-Depth Matrisome and Glycoproteomic Analysis of Human Brain Glioblastoma Versus Control Tissue

Glioblastoma (GBM) is the most common and malignant brain tumour. GBM is characterised by the aberrant activation of multiple receptor tyrosine kinase (RTK) signaling pathways. Heparan sulfate proteoglycans (HSPGs) and chondroitin sulfate proteoglycans (CSPGs), the most abundant component of brain extracellular matrix (ECM), have been reported to be involved in the regulation of many RTK signalling pathways and brain cancers. Proteoglycans (PGs) are consisted of a core protein and covalently attached linear polysaccharides known as glycosaminoglycans (GAGs). In this work, the researchers investigated the alterations of PGs, GAGs, as well as other constituents in the brain ECM in GBM versus control human brain tissue samples by performing label-free quantification analysis via liquid chromatography–tandem mass spectrometry (LC–MS/MS). Most proteins in the brain ECM were elevated in GBM tissue, including collagens, laminins, fibronectin, tenascins, fibrinogens, and various PGs, including CSPG4, agrin, biglycan, glypican 1, and prolargin, when compared to control samples. Five proteins, including tenascin R, hyaluronan proteoglycan link protein 1 and 2, decorin, and laminin subunit beta-2, were decreased in GBM tissue. In addition, increased 6-O CS sulfation and decreased HS 6-O sulfation were observed in GBM tissue relative to controls, accompanied by elevated unsulfated CS and HS disaccharides. Moreover, significant increases in glycosylation enzymes including glycosyltransferase and glycosidase were observed in GBM tissue when compared to controls. Taken together, these findings provided a better understanding of ECM changes in GBM pathogenesis and could facilitate the exploitation of novel makers and therapeutic targets.

How was PEAKS used?

Mass spectrometry data were collected from GBM and control tissue microarray cores. The data were searched using PEAKS DB and PEAKS PTM using PEAKS X+ against a Uniprot human database (homo sapiens) for protein identification. A precursor mass tolerance of 10 ppm, a fragment mass tolerance of 0.02 Da, and a maximum of 3 missed cleavages were applied. The variable modifications including hydroxylation P, oxidation M, hydroxylation K, hydroxylation-Hex K, hydroxylation Hex-Hex K, HexNAc ST, HexHexNAc ST, phosphorylation STY, ubiquitination K, deamidation N, methoxy K, and nitrotyrosine Y were added in PEAKS PTM. One unique peptide with at least two spectra and a false discovery rate (FDR) of less than 1% were applied for confident protein identification. Label free quantitation was performed using PEAKS Q with a mass error tolerance of 10 ppm and a retention time shift tolerance of 2.0 min.

Sethi, M. K., Downs, M., Shao, C., Hackett, W. E., Phillips, J. J., & Zaia, J. (2022). In-Depth Matrisome and Glycoproteomic Analysis of Human Brain Glioblastoma Versus Control Tissue. Molecular & Cellular Proteomics21(4). doi:10.1016/j.mcpro.2022.100216


Glioblastoma (GBM) is the most common and malignant primary brain tumor. The extracellular matrix, also known as the matrisome, helps determine glioma invasion, adhesion, and growth. Little attention, however, has been paid to glycosylation of the extracellular matrix components that constitute the majority of glycosylated protein mass and presumed biological properties. To acquire a comprehensive understanding of the biological functions of the matrisome and its components, including proteoglycans (PGs) and glycosaminoglycans (GAGs), in GBM tumorigenesis, and to identify potential biomarker candidates, we studied the alterations of GAGs, including heparan sulfate (HS) and chondroitin sulfate (CS), the core proteins of PGs, and other glycosylated matrisomal proteins in GBM subtypes versus control human brain tissue samples. We scrutinized the proteomics data to acquire in-depth site-specific glycoproteomic profiles of the GBM subtypes that will assist in identifying specific glycosylation changes in GBM. We observed an increase in CS 6-O sulfation and a decrease in HS 6-O sulfation, accompanied by an increase in unsulfated CS and HS disaccharides in GBM versus control samples. Several core matrisome proteins, including PGs (decorin, biglycan, agrin, prolargin, glypican-1, and chondroitin sulfate proteoglycan 4), tenascin, fibronectin, hyaluronan link protein 1 and 2, laminins, and collagens, were differentially regulated in GBM versus controls. Interestingly, a higher degree of collagen hydroxyprolination was also observed for GBM versus controls. Further, two PGs, chondroitin sulfate proteoglycan 4 and agrin, were significantly lower, about 6-fold for isocitrate dehydrogenase-mutant, compared to the WT GBM samples. Differential regulation of O-glycopeptides for PGs, including brevican, neurocan, and versican, was observed for GBM subtypes versus controls. Moreover, an increase in levels of glycosyltransferase and glycosidase enzymes was observed for GBM when compared to control samples. We also report distinct protein, peptide, and glycopeptide features for GBM subtypes comparisons. Taken together, our study informs understanding of the alterations to key matrisomal molecules that occur during GBM development.