Mapping phosphorylation of peptide biomarkers corresponding with glioblastoma tumours using data-dependent acquisition and multi-reflecting time-of-flight mass spectrometry

Significance of the Topic

Mapping phosphorylation events in glioblastoma cells addresses critical gaps in understanding tumor biology and treatment response. Phosphorylation regulates key signaling cascades, and reliable characterization of phosphopeptides can reveal pathways driving tumor progression and therapeutic resistance.

Objectives and Study Overview

This study aimed to develop and validate a liquid chromatography-mass spectrometry workflow for selective enrichment, identification, and localization of phosphorylation sites in peptide biomarkers derived from human glioblastoma cell lines. The approach combines data-dependent acquisition with a novel multi-reflecting time-of-flight mass spectrometer to achieve high resolution and accurate mass measurements.

Methodology

Four adult primary glioblastoma cell lines were cultured and treated with arginine deprivation therapy using ADI-PEG20. Treated and control cells were lysed with protease and phosphatase inhibitors, followed by reduction, alkylation, and tryptic digestion. Phosphopeptides were enriched using FeIII-NTA spin columns to improve detection of low-abundance species prior to mass spectrometric analysis.

Instrumentation

Liquid chromatography was performed on an ACQUITY Premier platform fitted with a Premier Peptide CSH Column (2.1 x 100 mm) at 45 °C using a 30-minute gradient from 1 to 35 percent acetonitrile with 0.1 percent formic acid, at 150 µL/min. Mass spectrometry was conducted on a Xevo MRT instrument operated in data-dependent acquisition mode, scanning m/z 50–2000 at 10 Hz for MS1 and 4 Hz for MS2, selecting the top 15 precursor ions per cycle, with dynamic exclusion of 30 seconds and collision energy ramps optimized for charge states 2+, 3+, and 4+.

Main Results and Discussion

Enrichment using FeIII-NTA columns enabled effective isolation of phosphopeptides, as evidenced by characteristic neutral losses of 98 Da in MS2 spectra. The Xevo MRT’s high resolution (>70,000 FWHM) and mass accuracy (<500 ppb) facilitated confident localization of phosphorylation sites and identification of over 320 phosphoproteins. Subsequent pathway analysis via MetaCore revealed altered networks related to kinase activation, cytoskeleton regulation, protein folding, and neurological processes. Treatment with arginine deprivation therapy induced distinct changes in phosphorylation profiles compared to controls, highlighting metabolic therapy effects on signaling pathways.

Benefits and Practical Applications

• High-resolution, rapid-scanning MS enables precise site localization of labile modifications.
• Selective phosphopeptide enrichment improves detection of low-abundance signaling molecules.
• Workflow adaptable for biomarker discovery, drug target validation, and quality control in clinical proteomics.

Future Trends and Applications

Advancements may include integration of data-independent acquisition strategies to complement targeted DDA workflows, deeper coverage of the phosphoproteome through optimized enrichment chemistries, and application to patient-derived samples for translational biomarker studies. Emerging high-throughput MS platforms may further streamline clinical proteomics pipelines for personalized oncology.

Conclusion

The combined use of FeIII-NTA enrichment, ACQUITY Premier chromatography, and Xevo MRT mass spectrometry provides a robust platform for comprehensive phosphoproteome profiling in glioblastoma. The method delivers high confidence in phosphorylation site assignment and yields insights into treatment-induced signaling alterations, supporting future therapeutic development.

References

• Poon M T C Sudlow C L M Figueroa J D et al Longer term ≥2 years survival in patients with glioblastoma in population based studies pre and post 2005 a systematic review and meta analysis Sci Rep 10 11622 2020 doi 10.1038 s41598 020 68011 4
• Qin A Musket A Musich P R Schweitzer J B Xie Q Receptor tyrosine kinases as druggable targets in glioblastoma Do signaling pathways matter Neuro Oncol Advances 3 1 vdab133 2021 doi 10.1093 noajnl vdab133
• Srinivasan A Sing J C Gingras A C Rost H L Improving phosphoproteomics profiling using data independent mass spectrometry J Proteome Res 21 8 1789 1799 2022 doi 10.1021 acs jproteome 2c00172
• Hajji N Garcia Revilla J Soto M S Perryman R Symington J Quarles C C Healey D R Guo Y Orta Vazquez M L Mateos Cordero S et al Arginine deprivation alters microglial polarity and synergizes with radiation to eradicate non arginine auxotrophic glioblastoma tumors J Clin Invest 132 6 2022 doi 10.1172 JCI142137