Preparation Of Neovascular Tissues from Human Glioma Tissues for Quantitative Proteomics Analysis of Tumor Angiogenesis

We perform proteomic profiling of glioma vasculature across tumor grades to identify biomarkers and neuropathic targets, reviving mechanisms driving angiogenesis and tumor progression. Existing methods face protein degradation in complete lysates, scarce grade one samples, and nonvascular contamination drawing LCM. This protocol optimizes handling and improves vascular protein profiling accuracy.

To begin, obtain polyethylene naphtholate membrane and reusable slides. Manually coat the membrane onto the reusable slide and store the coated slide at minus 80 degrees Celsius. Maintain the cryostat at minus 25 degrees Celsius for both the chamber and specimen head.

Retrieve glioma tissue from minus 80 degrees Celsius storage and transfer it on ice. Remove residual blood with filter paper. Apply embedding medium to a pre-cool disc and rapidly embed the tissue block.

Freeze the entire block at minus 25 degrees Celsius for 10 minutes to harden. After sectioning the tissue, mount the tissue sections onto a standard slide. Stain the sections with the 0.5%toluidine blue solution and screen neovascular vessels under an optical microscope.

Then transfer the vessel positive tissue section onto the polyethylene naphtholate coated laser, capture micro dissection slide. Seal the slide, label it, and store at minus 80 degrees Celsius. Fix the slide mounted tissue, stain it, and cryopreserve the processed sample at minus 80 degrees Celsius.

Start the laser capture microdissection instrument in advance and preheat for 30 minutes. Use the solid state infrared laser to preheat for another 30 minutes, and install a 500 microliter, micro-centrifuged tube collection device. Next, load the tissue sample mounted laser capture, micro dissection slide onto the prepared instrument, and adjust the focus on neovascular vessels in glioma tissues, using the compatible software.

Outline the laser marking line precisely along the outer edge of the neovascular vessel, while avoiding the vascular wall. Use the laser to burn and separate the neovascular vessel and collect the isolated vessel into the micro-centrifuged tube cap. Invert and seal the collected neovascular vessel sample tube and store the tube in a minus 80 degrees Celsius freezer.

Resuspend the collected tissue in the protein extraction buffer. Sonicate the suspension in an ice water bath for one minute to achieve complete lysis. Centrifuge the lysate at 16, 000 G for 10 minutes, and transfer the supernatant to a new micro-centrifuged tube.

Aliquot a minimal volume of the supernatant for protein quantification, using the by synkinetic acid assay. Add dithioerythritol to the remaining supernatant to a final concentration of one millimolar, and mixed gently by pipetting to avoid bubble formation. After aliquoting the treated supernatant, store at minus 80 degrees Celsius.

Add ice cold acetone to each extracted protein sample at a sample to acetone ratio of one to five volume per volume. After mixing, incubate the sample at minus 20 degrees Celsius for one hour, and then centrifuge at 12, 000 G at four degrees Celsius for 15 minutes. Discard the supernatant to remove sodium dodecyl sulfate, dithioerythritol, and tris-hydrochloride.

Re-suspend the pellet in 20 microliters of dissolution buffer containing one microliter of 1%sodium dodecyl sulfate. Next, add two microliters of dithioerythritol reducing reagent to the sample and mix thoroughly. Incubate at 60 degrees Celsius for one hour, then add one microliter of iodoacetamide cysteine blocking reagent and incubate at room temperature for 10 minutes.

Add trypsin to each sample at a protein to trypsin ratio of 20 to one volume per volume. Vortex mix the sample and incubate it at 37 degrees Celsius overnight. Reconstitute each iTRAQ sample with 50 microliters of isopropanol.

Add the corresponding triptych peptide sample to the reagent tube, and incubate at room temperature for one hour. After incubation, add three times the reaction volume of ultrapure water to terminate the reaction, and incubate at room temperature for 30 minutes. Equally pool six labeled samples, mix thoroughly, and lyophilize.

Reconstitute the lyophilized peptides in two milliliters of solution A, containing 10 millimolar, potassium dihydrogen phosphate, and 25%acetonitrile at pH 2.7. After running the sample through a sulfoethyl functionalized cation exchange column, collect fractions using suitable buffer gradients into separate tubes. Finally, concentrate each fraction by vacuum centrifugation.

Reconstitute each fraction with 40 microliters of 0.1%volume per volume trichloroacetic acid, and store all fractions at minus 80 degrees Celsius. Perform LCMSMS to analyze each fractionated peptide sample. The mass deviation of identified peptides followed a normal distribution, centered around zero.

The peptide length distribution showed that the vast majority of peptides had lengths of eight to 20 amino acid residues. The distribution of relative mass of all identified proteins shows that almost 100%of the protein molecular weights are distributed between zero to 200 kilodaltons. And most of the protein isoelectric points are distributed between four to 10.

Quantitative analysis identified 371 differentially expressed proteins in glioma neovascular tissues compared to controls, including 170 upregulated proteins and 201 downregulated proteins. Gene ontology analysis identified 76 statistically significant cellular components, 75 statistically significant biological processes, and 46 statistically significant molecular functions among the 371 differentially expressed proteins. Kyoto Encyclopedia of genes and genomes pathway analysis identified 23 statistically significant signaling pathway changes among the 371 differentially expressed proteins.

Protein protein interaction network analysis identified 25 hub differentially expressed proteins, using four algorithms. This protocol enables, analyze our differential protein expression in glioma vasculature, identifying grade specific angiogenesis related proteins and quantifying protein omic genes, using air tract technology. An important challenge is to locate tumor neovascularization under an optical microscope and collect the dissected vessels accurately.

Our future studies will analyze grade specific angiogenesis protein omics, PTM omics, pathway harps, and multi omics integrated biomarkers and drug targets.