MAPPING PHOSPHORYLATION OF PEPTIDE BIOMARKERS CORRESPONDING WITH GLIOBLASTOMA TUMOURS USING DDA AND A NOVEL MRT MASS SPECTROMETER

Importance of the topic

Glioblastoma represents one of the most aggressive primary brain malignancies, characterized by poor prognosis and limited therapeutic options. Phosphorylation plays a central role in regulating enzyme and kinase activity within cancer signaling pathways. Mapping site-specific phosphorylation events in glioblastoma can reveal biomarkers and therapeutic targets, supporting the development of precision treatments and improving our understanding of tumor cell response to metabolic interventions.

Objectives and Overview of the Study

This study aimed to profile phosphorylation changes in human glioblastoma cell lines subjected to arginine deprivation therapy using the enzyme ADI-PEG20. Leveraging a novel benchtop high-resolution mass spectrometer (Xevo MRT MS) and data dependent acquisition (DDA), the work sought to enrich, identify, and localize phosphopeptides before and after treatment. Network analysis was performed to interpret pathway alterations linked to metabolic therapy.

Methodology and Instrumentation

Four adult primary glioblastoma cell lines were cultured and either treated with ADI-PEG20 or left untreated. Cells were lysed in a buffer containing protease and phosphatase inhibitors. Protein extracts underwent reduction, alkylation, and tryptic digestion. Phosphopeptides were selectively enriched using Fe(III)-NTA spin columns to overcome low abundance and labile phosphorylation challenges.

Liquid chromatography was performed on an ACQUITY Premier UPLC system equipped with a Peptide CSH column (2.1 x 100 mm) at 45 °C. A 30-minute gradient from 1 to 35% acetonitrile (0.1% formic acid) was applied at 150 µL/min.

Mass spectrometry was carried out on a Xevo MRT MS platform operated in DDA mode. Survey scans covered 50–2000 m/z at 10 Hz, followed by MS/MS of the top 15 ions at 4 Hz. The instrument delivered mass resolution >70,000 FWHM and mass accuracy <500 ppb. Data processing and phosphosite localization were performed with ProteinLynx Global SERVER (PLGS).

Main Results and Discussion

• Enrichment using Fe(III)-NTA spin columns enabled selective recovery of phosphopeptides and removal of unmodified peptides.
• DDA on the Xevo MRT MS provided high-quality MS/MS spectra, allowing confident localization of phosphorylated sites.
• Over 320 phosphorylated proteins were identified across treated and untreated samples.
• Pathway analysis with MetaCore revealed alterations in cytoskeleton regulation, neurological signaling (e.g., LRRK2 pathways), translation, and protein folding networks in response to arginine deprivation.
• Metabolic therapy induced distinct phosphorylation profiles, highlighting activation of kinase-driven pathways and changes in cytoskeletal and neuronal protein regulation.

Benefits and Practical Applications

• The combined phosphopeptide enrichment and high-resolution DDA workflow enables comprehensive profiling of low-abundance PTMs.
• Xevo MRT MS offers rapid scanning and exceptional mass accuracy for unambiguous peptide identification.
• The approach supports biomarker discovery and evaluation of metabolic therapies in cancer research.

Future Trends and Applications

Further studies will focus on elucidating the downstream cellular effects of altered phosphorylation networks under metabolic stress. Integration with systems biology and expanded PTM analyses may deepen insights into treatment mechanisms. Adoption of this workflow for clinical sample profiling and other post-translational modifications is anticipated to enhance translational research in oncology.

Conclusion

This work demonstrates an effective LC-MS workflow combining phosphopeptide enrichment and high-resolution DDA on the Xevo MRT MS. It successfully mapped phosphorylation changes induced by arginine deprivation in glioblastoma cell lines. The results provide a foundation for mechanistic studies of metabolic therapies and highlight promising pathways for targeted interventions.

Reference

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