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Integrated proteomics and N-glycoproteomic characterization of glioblastoma multiform revealed N-glycosylation heterogeneities and elevations in mannose, sialyation and fucosylation

ABSTRACT: Glioblastoma multiforme (GBM) is a highly malignant primary brain tumor. While tremendous efforts in multi-omics research to profile the dysregulated molecular mechanisms and cellular pathways of the disease have been exerted, the glycoproteomics of GBM remains incompletely understood. Glycosylation, as one of the most important post-translational modifications, is crucial in regulating cell proliferation and relevant oncogenic pathways. In this study, we systematically profiled the N-glycoproteomics of para-cancerous and cancerous tissues from GBM patients to reveal site-specific N-glycosylation patterns defined by intact glycopeptides (IGPs). A total of 1863 distinct IGPs with 161 N-linked glycan compositions and 326 glycosites were identified and quantified. We then discovered that 396 IGPs from 43 glycoproteins differed between adjacent and GBM tissues. A more in-depth characterization of the enriched functions of differentially expressed glycopeptides was performed. Thereafter, the proteomics and glycoproteomics data were combined, and the proportion of normalized glycosylation alterations was calculated to determine whether the difference in expression of glycopeptides could be attributed to global protein levels or glycosylation. Alterations in glycosylation triggered by site-specific N-glycans and glycoprotein abundance, as well as glycosite heterogeneity, were demonstrated. Ultimately, examination of the overall glycosylation levels revealed the positive contribution of oligo-mannose resulting from altered glycol-enzyme expression levels, along with aberrant complex glycans caused by increased sialylated or/and fucosylated glycans. Overall, the dataset highlights N-glycosylation heterogeneities and altered glycans at post-translational levels and provides valuable information for novel therapeutic approaches and specific detection strategies.

ORGANISM(S): Homo Sapiens

SUBMITTER: Huijuan Wang

PROVIDER: PXD029920 | iProX | Fri Nov 26 00:00:00 GMT 2021

REPOSITORIES: iProX

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Project description:Glycobiology plays a central role in prostate cancer (PCa), as evidenced by the aberrant glycosylation in PCa-derived glycoproteins. Understanding the protein glycosylation changes underling PCa onset and progression may open opportunities for the discovery of novel biomarkers for PCa detection and stratification as well as targets for PCa metastasis. Advances in mass spectrometry-based glycomics and glycoproteomics have opened up exciting avenues for deep characterisation of the immensely, yet poorly understood complex glycoproteome. In this study, we have used integrated glycomics and glycoproteomics to explore the protein, cell and tumour-grade specific N- and O- glycosylation signatures in surgically-removed fresh PCa tissues grouped into five PCa disease grades and tissues from benign prostatic hyperplasia patients (BPH). Porous graphitised carbon–liquid chromatography (PGC–LC)-MS/MS analysis was used to profile the N-and O-glycome, revealing PCa grade specific N and O glycosylation changes, including oligomannosidic and paucimannosidic, bisecting-GlcNAc and highly branched tri- and tetra-antennary fucosylated and sialylated N-glycans as well as sialylated core 1 and 2 type O-glycans. High resolution LC-MS/MS was then employed to capture the micro and macroheterogeneity of 1,085 N-glycosites (>7,400 unique N-glycopeptides) from 540 N-glycoproteins and 360 O-glycosites (>500 unique O-glycopeptides) from 178 O-glycoproteins and reveal protein and cell specific N- and O- glycosylation changes associated with PCa progression. We also showed PCa grade-specific increase in the expression of oligosaccharyltransferase (OST) subunits that correlate with changes in the site occupancy of N-glycoproteins. In conclusion, this study shows that integrated glycomics and glycoproteomics can provide unprecedented insight into key molecular features and site-specific glycan heterogeneity contributing to the highly diverse tumour-microenvironment and allow us to deconstruct the regulation of the protein, cell and tumour-grade-specific glycosylation associated with PCa onset and development.

Project description:Changes in IgG glycosylation have been reported to be associated with colorectal cancer (CRC), but the alteration in specific subclass of IgG is unknown. Herein, we optimized five common IgG glycopeptides enrichment methods to acquire comprehensive profiling of IgG glycoproteomics in CRC. Notably, incomplete tryptic digestion of IgG occurred when using ordinary ratio of protease to protein, which significantly impacted the final statistical analysis of association between IgG glycosylation and CRC. We introduced a method based on two-step enzymatic digestion, enabling the complete digestion of IgG glycopeptides and further improving the detection intensity of the target glycopeptides. By this approach, total 47 IgG glycopeptides can be identified by nanoLC-ESI-MS/MS. Following rapid and automatic data processing through the self-developed program, we observed that IgG1_H3N4F1 and IgG1_H3N4 were substantially increased in CRC, while IgG1_H5N5F1, IgG1_H5N4F1S1and IgG2_H5N4F1 were markedly decreased. Strikingly, these four glycopeptides were continually significantly changed in early and late stages of CRC. Further evaluation of the diagnostic performance showed they all obtained a fair performance in discriminating the patients from the normal. In terms of the glycan features, it demonstrated that CRC progression associates with the increased agalactosylation, and the decreased digalactosylation and galactosylation per antenna on diantennay glycans of IgG1 and IgG2. Concurrently, the decreased sialylation of IgG1 was strongly correlated with CRC. Moreover, tumor-specific glycosylation analysis showed that the alterations of subclass-specific IgG glycosylation were more significant in colon cancer, and no obvious difference between colon and rectal cancer was found. More importantly, all these findings were validated in an independent cohort. This two-step enzymatic approach is crucial to explore the relationship between the disease progression and IgG glycosylation.

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Integrated proteomics and N-glycoproteomic characterization of glioblastoma multiform revealed N-glycosylation heterogeneities and elevations in mannose, sialyation and fucosylation

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