Figure legends:
Figure 1. An overview of the study and schematic of
methodological workflow. A) Glioma tissue samples were collected from
11 high grade glioma patients and 5 low grade glioma patients. After the
lysate preparation and protein digestion, label free quantification was
performed. B) The raw files generated from the mass spectrometer was
analysed using MaxQuant followed by statistical data analysis. C)
Significant proteins were selected from the global proteomics data and
multiple reaction monitoring assay was performed for validation. D)In-silico molecular docking of target proteins was performed to
identify the potential drug candidates.
Figure 2. Quantitative proteomic profiling and pathway
enrichment analysis of glioma samples. A) Heatmap of top 25
differentially expressed proteins in HGG and LGG. B) PCA plot showing
two separate clusters for HGG and LGG with a score of 33% for PC1,
13.3% for PC2 and 10.1% for PC3. C) Graphical representation of the
significance in the differential expression was quantitatively performed
using volcano plot: log10 (p-value) vs.
log2 (fold change). D) Gene set enrichment analysis
revealed positively and negatively enriched pathways. E) Epithelial
Mesenchymal Transition pathway is found to be highly enriched pathways
in HGG with normalized enrichment score 1.77. F) Oxidative
phosphorylation process is found to be highly enriched pathways in LGG
with normalized enrichment score -1.87.
Figure 3. Protein-protein interaction analysis of significant
proteins in high grade gliomas and their mapped pathways. Focal
adhesion, ECM receptor interaction, N-Glycan biosynthesis and Complement
coagulation cascade pathways are found to be highly upregulated in HGGs.
Violin plots showing differentially expressed proteins in HGG and LGG
(**1.00e-03 < p ≤ 1.00e-02; ***1.00e-04 < p ≤
1.00e-03; ****p ≤ 1.00e-04).
Figure 4. Validation of significant proteins by
multiple reaction monitoring assay. A) each sample was spiked with
fixed amount of heavy labelled peptide and the Coefficient of variance
is 10%. RPN1 and RPN2 are the subunits of oligosaccharyltransferase
(OST). Box plots showing differentially expressed proteins in HGG and
LGG (**1.00e-03 < p ≤ 1.00e-02; ***1.00e-04 < p ≤
1.00e-03).
Figure 5. In-silico molecular docking of catalytic
subunits of oligosaccharyltransferase complex with FDA approved drugs.A) The 3D representation of predicted binding pocket of Entrectinib. The
binding affinity of Entrectinib with STT3A is -10.7 kcal/mol and with
STT3B is -10.6 kcal/mol. B) The 3D representation of predicted binding
pocket of Irinotecan. The binding affinity of Irinotecan with STT3A is
-11 kcal/mol and with STT3B is -10.6 kcal/mol. C) The predicted 2D
interaction map of Entrectinib with STT3A. D) The predicted 2D
interaction map of Irinotecan with STT3A.
Figure 6. Molecular mechanism associated with OST
mediated aggressive behaviour of high-grade gliomas. Malignant
transformation of glioma cells involves extra-cellular matrix
remodelling which is accompanied by OST mediated aberrant glycosylation.
Glycosylation also results in activation of TGF- β signalling pathways.
TGF- β is a strong inducer of EMT transcription factors, drives the EMT
in both SMAD and non-SMAD signalling pathways.