High-grade gliomas (HGGs) are extremely aggressive primary brain tumors with high mortality rates. Despite notable advancements in clinical research, HGGs are still characterized by poor prognosis and appalling survival rates. Several global proteomics studies have reported many protein biomarkers however, the transition of these proteins biomarkers from lab to clinic is exceptionally low. In the current study, over-expressed HGG proteins identified from various global proteomics studies were validated in HGG tissue samples to explore their role as potential therapeutic candidates.

Radiologically and histopathologically confirmed high-grade glioma tissue samples were collected after the surgery. All the patients participated in the study had provided their written consent before surgery. For MS analysis, tissue lysates were prepared in 8 M urea buffer containing Tris, NaCl and MgCl2. Tissue lysis was performed by sonicating the tissue at 40% amplitude for 2.5 min, further to ensure the complete tissue lysis bead beating was performed to homogenize the tissue. The homogenized tissue lysate was centrifuged around 6000 x g for 15 min at 4°C and supernatant was collected. Prior to the protein digestion, protein quantification was performed by Bradford assay and approximately 30 µg of protein was used for digestion. First, 20 mM Tris (2-carboxyethyl) phosphine (TCEP) was added to reduce the disulfide bonds, followed by addition of 40 mM iodoacetamide (IAA) for alkylation. Finally, trypsin was added at an enzyme/substrate ratio of 1:50 and kept the tubes at 37°C for overnight digestion. Next, digested samples were desalted using C18 stage tips. The Transition list was prepared by importing the proteins in skyline daily version 22.2.1.501. Only unique peptides with 8 to 20 amino acids length were selected and missed cleavage criteria was set to 0. Precursor charge and product charge was selected as +2, +1 respectively and all y ions from ion 1 to last ion -2 were monitored. Unique peptides from SRM atlas were also selected and imported in skyline daily during transition list preparation. The run was carried out on a TSQ Altis mass spectrometer (ThermoFisher Scientific, USA) coupled to a Vanquish uHPLC (ThermoFisher Scientific, USA) platform. For each run, 1.5 µg of peptides were loaded on to a Hypersil Gold C18 column 1.9mm 100 X 2.1mm (ThermoFisher Scientific, USA). A binary buffer system consisting of Buffer A (0.1% Formic acid in water) and Buffer B (80% Acetonitrile in 0.1% Formic acid water) was used for chromatographic separation of peptides with flow rate of 0.45 ml/min, keeping the total gradient time of 15 min. The obtained data was analyzed using Skyline daily version 20.2.1.501. To visualize the data PROSIT spectral library was used and statistical data analysis was performed using MSstat tool. All the data and results have been made available here.

The sample details included in the study is mentioned below: Control1: Temporal region Control2: Occipital Pole region Control3: Frontal region Control4: Occipital Pole region Control5: Temporal region Control6: Parietal region Control7: Frontal region Control8: Temporal region Control9: Parietal region Control10: Frontal region Control11: Parietal region Control12: Frontal region Control13: Occipital Pole region Control14: Temporal region Control15: Occipital Pole region Control16: Parietal region HGG mut1: Grade IV, IDH mut HGG mut2: Grade IV, IDH mut HGG mut3: Grade IV, IDH mut HGG mut4: Grade IV, IDH mut HGG mut5: Grade IV, IDH mut HGG mut6: Grade IV, IDH mut HGG mut7: Grade IV, IDH mut HGG mut8: Grade IV, IDH mut HGG mut9: Grade IV, IDH mut HGG mut10: Grade IV, IDH mut HGG mut11: Grade III, IDH mut HGG mut12: Grade III, IDH mut HGG wt1: Grade IV, IDH wt HGG wt2: Grade IV, IDH wt HGG wt3: Grade IV, IDH wt HGG wt4: Grade IV, IDH wt HGG wt5: Grade IV, IDH wt HGG wt6: Grade IV, IDH wt HGG wt7: Grade IV, IDH wt HGG wt8: Grade IV, IDH wt HGG wt9: Grade IV, IDH wt HGG wt10: Grade IV, IDH wt HGG wt11: Grade IV, IDH wt HGG wt12: Grade IV, IDH wt HGG wt13: Grade IV, IDH wt HGG wt14: Grade IV, IDH wt HGG wt15: Grade IV, IDH wt HGG wt16: Grade IV, IDH wt HGG wt17: Grade IV, IDH wt HGG wt18: Grade IV, IDH wt HGG wt19: Grade IV, IDH wt HGG wt20: Grade IV, IDH wt HGG wt21: Grade IV, IDH wt HGG wt22: Grade IV, IDH wt HGG wt23: Grade IV, IDH wt HGG wt24: Grade IV, IDH wt HGG wt25: Grade IV, IDH wt HGG wt26: Grade IV, IDH wt HGG wt27: Grade IV, IDH wt HGG wt28: Grade IV, IDH wt HGG wt29: Grade IV, IDH wt HGG wt30: Grade IV, IDH wt HGG wt31: Grade IV, IDH wt HGG wt32: Grade IV, IDH wt HGG wt33: Grade IV, IDH wt HGG wt34: Grade IV, IDH wt HGG wt35: Grade IV, IDH wt HGG wt36: Grade IV, IDH wt HGG wt37: Grade IV, IDH wt HGG wt38: Grade IV, IDH wt HGG wt39: Grade IV, IDH wt HGG wt40: Grade IV, IDH wt HGG wt41: Grade IV, IDH wt HGG wt42: Grade IV, IDH wt HGG wt43: Grade IV, IDH wt HGG wt44: Grade IV, IDH wt HGG wt45: Grade IV, IDH wt HGG wt46: Grade IV, IDH wt HGG wt47: Grade IV, IDH wt HGG wt48: Grade IV, IDH wt HGG POOL1: Pool sample for QC HGG POOL2: Pool sample for QC HGG POOL3: Pool sample for QC HGG POOL4: Pool sample for QC HGG POOL5: Pool sample for QC HGG POOL6: Pool sample for QC HGG POOL7: Pool sample for QC HGG POOL8: Pool sample for QC HGG POOL9: Pool sample for QC HGG POOL10: Pool sample for QC