Global In-Depth Quantitative Phosphoproteomic Analysis of HIV Infected Cells using the Orbitrap Fusion Tribrid Mass Spectrometer

Importance of the Topic

Phosphorylation mediated signaling controls many aspects of the cellular response to viral infection including immune defense and cell cycle regulation. A detailed quantitative map of phosphorylation dynamics in HIV infected cells can reveal key host kinases and substrates at the virus human interface and support therapeutic target discovery.

Objectives and Study Overview

The study aimed to globally identify and quantify changes in the host phosphoproteome of SILAC labeled Jurkat T cells following infection with HIV strain NL4-3. By comparing infected and mock infected cells this work sought to define signaling pathways and phosphorylation events induced by HIV.

Methodology and Applied Instrumentation

Sample preparation employed stable isotope labeling with amino acids in cell culture and HILIC TiO2 enrichment of phosphopeptides. Peptides were separated on a nanoflow liquid chromatography system composed of EASY-nLC 1000 with EASY-Spray source and a long analytical column. Mass spectrometry analysis used the Orbitrap Fusion Tribrid instrument with a decision tree driven workflow that interleaves high resolution Orbitrap full scans with either collisional induced dissociation or electron transfer dissociation scans based on precursor charge characteristics. Data analysis was performed with Proteome Discoverer and the PhosphoRS module for confident site localization.

Main Results and Discussion

• The combined ETD CID fragmentation approach identified significantly more unique phosphopeptides than CID alone and improved localization of phosphate sites even in multiply phosphorylated peptides
• High resolution full scan data acquired in parallel with MS MS fragmentation enabled accurate SILAC quantitation along with sensitive phosphopeptide identification
• Phosphorylation profiling revealed activation of aurora kinases upon HIV infection correlating with G2 M cell cycle arrest and suggesting that HIV proteins VIF and VPR modulate the DNA damage response pathway

Benefits and Practical Applications

Utilizing the Tribrid architecture and dynamic fragmentation strategy enhances both throughput and depth of phosphoproteomic coverage. This approach provides a robust platform for systematic analysis of signaling networks in infection biology quality control applications and targeted biomarker discovery.

Future Trends and Potential Uses

Further development of adaptive data acquisition strategies and integration of advanced software tools will enable even deeper coverage of phosphorylation events. Expansion to primary clinical samples and other viral systems may uncover universal and pathogen specific signaling responses supporting new antiviral interventions.

Conclusion

The Orbitrap Fusion Tribrid mass spectrometer combined with an intelligent ETD CID decision tree yields a powerful workflow for in depth quantitative phosphoproteomics. Applied to HIV infected cells this strategy revealed critical kinase activation events offering insights into viral pathogenesis and potential targets for therapeutic development.

Reference

1. Swaney DL McAlister GC Coon JJ Nat Methods 2008 5 959
2. Taus T Kocher T Pichler P et al J Proteome Res 2011 10 5354
3. Beltrao P Albanèse V Kenner LR et al Cell 2012 150 413
4. Sakai K Dimas J Lenardo MJ PNAS USA 2006 103 3369