TITLE: Profiling the Surfaceome of Meningioma for Immunotherapeutic Target Identification
AUTHORS: ABDULLAH BIN ZUBAIR, Muhammad Awais Khan, Fatima Hamid, Renee M. Hirte, mlabib, dgandhi

[DESCRIPTION]
This protocol aims to profile the surfaceome of meningioma tumors and matched healthy tissue to identify differentially expressed tumor cell-surface proteins and tumor-specific antigens. By using N-Glycocapture Mass Spectrometry, we will enrich and analyze the cell surface glycoproteins, allowing for the identification of potential immunotherapeutic targets. The protocol includes sample collection, tissue processing, N-Glycocapture enrichment, Mass Spectrometry analysis, and data interpretation. We anticipate that this protocol will provide valuable insights into the surfaceome of meningioma cells, enhancing our understanding of tumor biology and facilitating the development of precise, targeted immunotherapies.

[STEPS]
SECTION: Subject Terms:
1. Meningioma, Immunotherapy, Surfaceome profiling, Target identification
SECTION: Keywords:
2. Meningioma, Immunotherapy, Surfaceome, Proteomics, Mass Spectrometry
SECTION: Introduction
3. Meningioma is the most common primary brain tumor in adults, characterized by significant treatment challenges and variable recurrence rates. Immunotherapy has demonstrated promise in other cancer types, but the lack of specific cell surface targets unique to meningioma hampers its application in this context. This protocol aims to overcome this challenge by profiling the surfaceome of meningioma cells, identifying differentially expressed tumor-specific antigens, and validating their functional significance as potential immunotherapeutic targets.
SECTION: Reagents and Equipment:
4. - N-Glycocapture Kit (Manufacturer: Glycopath. Inc , Catalog Number: Basic N-Glycan Imaging Kit)
- Mass Spectrometer (Manufacturer: Bruker, Model: TimsTOF Pro 1, Mass Analyzer: IonMob.-Q-Time-Of-Flight (IM-Qq-TOF), m/z Range: 50-40'000, LC-Detector: MS only, Chromatographic Separation: e.g. nano C18, Sample types: Proteomics, Peptides, Proteins, Digest (Sequence identification), Operation Modes: MS, MS/MS (CID, PASEF).
SECTION: Procedure
6. Tissue collection and processing
SECTION: Procedure
6.1. Obtain meningioma tumor tissue and matched healthy tissue during surgical resection.
SECTION: Procedure
6.2. Immediately transfer the collected tissue samples to a sterile container filled with ice-cold phosphate-buffered saline (PBS) to maintain tissue integrity.
SECTION: Procedure
6.3. Label each container with the patient's unique identifier and the type of tissue (tumor or healthy) using a waterproof marker.
SECTION: Procedure
6.4. Transport the tissue samples to the laboratory on ice within 30 minutes of collection.
SECTION: Procedure
7. Tissue preparation and homogenization:
SECTION: Procedure
7.1. In a sterile laminar flow hood, transfer each tissue sample to a pre-chilled petri dish.
SECTION: Procedure
7.2. Using a sterile scalpel, remove any visible connective tissue or blood vessels from the tissue samples.
SECTION: Procedure
7.3. Cut the tissue into small pieces (~1-2 mm) using sterile scissors and forceps.
SECTION: Procedure
7.4. Transfer the tissue pieces to pre-labeled microcentrifuge tubes containing ice-cold lysis buffer provided in the N-Glycocapture Kit.
SECTION: Procedure
7.5. Ensure that the tissue-to-lysis buffer ratio is appropriate (recommended ratio: 1:10) and adjust as necessary.
SECTION: Procedure
7.6. Homogenize the tissue samples using a tissue homogenizer or a motorized pestle until a homogeneous suspension is achieved.
SECTION: Procedure
7.7. Centrifuge the homogenized samples at 10,000 x g for 10 minutes at 4°C to pellet cellular debris and collect the supernatant containing the solubilized proteins.
 
SECTION: Procedure
8. N-Glycocapture enrichment
SECTION: Procedure
8.1. Add the appropriate volume of N-Glycocapture beads to the collected supernatant based on the manufacturer's instructions.
SECTION: Procedure
8.2. Incubate the sample-bead mixture on a rotator or shaker at 4°C for 2 hours to allow for the specific binding of glycoproteins to the beads.
SECTION: Procedure
8.3. Place the sample-bead mixture on a magnet or use a magnetic separator to separate the beads from the supernatant.
SECTION: Procedure
8.4. Carefully remove the supernatant without disturbing the beads and discard it.
SECTION: Procedure
8.5. Wash the beads three times with ice-cold wash buffer provided in the N-Glycocapture Kit, following the manufacturer's instructions.
SECTION: Procedure
8.6. After the final wash, resuspend the beads in the elution buffer provided in the N-Glycocapture Kit to release the captured glycoproteins.
SECTION: Procedure
8.7. Incubate the resuspended beads on a rotator or shaker at room temperature for 10 minutes to elute the glycoproteins.
SECTION: Procedure
8.8. Separate the beads from the eluted glycoproteins using a magnet or magnetic separator and collect the glycoprotein-containing supernatant.
SECTION: Procedure
9. Mass Spectrometry analysis
SECTION: Procedure
9.1. Transfer the collected glycoprotein-containing supernatant to a fresh microcentrifuge tube.
SECTION: Procedure
9.2. Prepare the sample for Mass Spectrometry analysis following the instrument manufacturer's guidelines, including protein digestion and peptide purification steps.
SECTION: Procedure
9.3. Load the purified peptides onto a Mass Spectrometer for data acquisition.
SECTION: Procedure
9.4. Analyze the acquired data using appropriate software to identify and quantify differentially expressed tumor cell-surface proteins and tumor-specific antigens.
SECTION: Troubleshooting
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SECTION: Time Taken
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SECTION: Anticipated Results
12. We anticipate identifying differentially expressed tumor cell-surface proteins and tumor-specific antigens in meningioma samples compared to healthy tissue. These findings will provide valuable insights into potential immunotherapeutic targets for precise, targeted treatments.
SECTION: References
13. 1. WHO Classification of Tumors Editorial Board. Central nervous System tumors. Lyon (France): International Agency for Research on Cancer; 2021. (WHO Classification of tumors series, 5th ed.; vol. 6).
 
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3. Alessia Pellerino, Bruno F, Palmiero R, Edoardo Pronello, Bertero L, Riccardo Soffietti, et al. Clinical Significance of Molecular Alterations and Systemic Therapy for Meningiomas: Where Do We Stand? Cancers [Internet]. 2022 Apr 30;14(9):2256–6. Available from: [https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9100910/](https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9100910/)
 
4. Bakhshinyan D, Suk Y, Kuhlmann L, Adile AA, Vladimir Ignatchenko, Custers S, et al. Dynamic profiling of medulloblastoma surfaceome. Acta neuropathologica communications. 2023 Jul 10;11(1).
 
5. Ferguson ID, Patiño-Escobar B, Tuomivaara ST, Lin YHT, Nix MA, Leung KK, et al. The surfaceome of multiple myeloma cells suggests potential immunotherapeutic strategies and protein markers of drug resistance. Nature Communications [Internet]. 2022 Jul 15 [cited 2023 Jul 9];13(1):4121. Available from: [https://www.nature.com/articles/s41467-022-31810-6](https://www.nature.com/articles/s41467-022-31810-6)
 
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7. Terabe M, Wu J. Rethinking Immunotherapy in Meningiomas. Neuro-Oncology. Volume 23, Issue 11, November 2021, Pages 1812–1813. [https://doi.org/10.1093/neuonc/noab168](https://doi.org/10.1093/neuonc/noab168)
 
8. Medici G, Freudenmann LK, Velz J, Sophie Shih-Yüng Wang, Konstantina Kapolou, Nagarajan Paramasivam, et al. A T-cell antigen atlas for meningioma: novel options for immunotherapy. Acta neuropathologica. 2023 June 27;146(2):173–90.
 
9. Pan S, Chen R, Aebersold R, Brentnall TA. Mass Spectrometry Based Glycoproteomics—From a Proteomics Perspective. Molecular & Cellular Proteomics. 2010 Aug 24;10(1):R110.003251.
 
10. Apweiler R, Hermjakob H, Sharon N. On the frequency of protein glycosylation, as deduced from analysis of the SWISS-PROT database. Biochimica et Biophysica Acta (BBA) - General Subjects [Internet]. 1999 Dec 17;1473(1):4–8. Available from: [https://www.sciencedirect.com/science/article/abs/pii/S0304416599001658?via%3Dihub](https://www.sciencedirect.com/science/article/abs/pii/S0304416599001658?via%3Dihub)
 
11. Justin Z Wang, Alexander P Landry, David R Raleigh, Felix Sahm, Kyle M Walsh, Roland Goldbrunner, Leeor S Yefet, Jörg C Tonn, Chloe Gui, Quinn T Ostrom, Jill Barnholtz-Sloan, Arie Perry, Yosef Ellenbogen, C Oliver Hanemann, Gerhard Jungwirth, Michael D Jenkinson, Ghazaleh Tabatabai, Tiit I Mathiesen, Michael W McDermott, Marcos Tatagiba, Christian la Fougère, Sybren L N Maas, Norbert Galldiks, Nathalie L Albert, Priscilla K Brastianos, Felix Ehret, Giuseppe Minniti, Katrin Lamszus, Franz L Ricklefs, Jens Schittenhelm, Katharine J Drummond, Ian F Dunn, Omar N Pathmanaban, Aaron Cohen-Gadol, Erik P Sulman, Emeline Tabouret, Emelie Le Rhun, Christian Mawrin, Jennifer Moliterno, Michael Weller, Wenya (Linda) Bi, Andrew Gao, Stephen Yip, Maximilian Niyazi, The International Consortium on Meningiomas (ICOM), Kenneth Aldape, Patrick Y Wen, Susan Short, Matthias Preusser, Farshad Nassiri, Gelareh Zadeh, Meningioma: International Consortium on Meningiomas (ICOM) consensus review on scientific advances & treatment paradigms for clinicians, researchers, and patients, Neuro-Oncology, 2024;, noae082. [https://doi.org/10.1093/neuonc/noae082](https://doi.org/10.1093/neuonc/noae082)
 
SECTION: Acknowledgments
14. We acknowledge the Colon Rectal Surgery Department at Mayo Clinic Rochester where the idea was in its budding phases and the process of individualized immunotherapy was considered a viable option for the future of cancer treatment plans.  Dr. David W. Larson M.D., M.B.A.,(PI) and Dr. Kellie L. Mathis, M.D. (PD CRS MAYO CLINIC-RST) were the supporting group that provided vast opportunities to their researchers to make better contributions to the field of medicine. 
ETH ZURICH the MS service at LOC is open to all research groups within ETH Zurich, provided the Individual, customer-specific development of fully automated processing scripts for large series of experiments (screenings), and provided knowledge and expertise to our customers as integral consulting around analytical questions with focus on mass spectrometry.  
SECTION: Guidelines and Warnings
5. This protocol needs prior approval by the users' institutional review board (IRB) or equivalent ethics committee(s). Before proceeding with this protocol, ensure that you have obtained the necessary ethical approval. By seeking ethical approval, you demonstrate your commitment to conducting research responsibly and ensuring the protection of participants' rights and well-being. This protocol is still pending process.