Examining the Structural Influence of Site-Specific Phosphorylation by Ion Mobility Mass Spectrometry

Significance of the Topic

Phosphorylation regulates protein structure and function in many cellular processes. Precise mapping of phosphorylation sites and understanding their conformational effects are crucial for basic research, drug development, and biomarker discovery. Traditional LC–MS approaches can struggle with low‐abundance phosphopeptides, inefficient ionization, and limited ability to distinguish positional isomers. Integrating ion mobility separation with mass spectrometry (IMS-MS) enhances the characterization of peptide conformers, improves site localization, and enables collisional cross section (CCS) measurements for structural insights.

Objectives and Overview of the Study

This work presents an automated end-to-end workflow—combining on-cartridge phosphopeptide enrichment, nano-LC, and ion mobility Q-TOF analysis—to:

• Efficiently enrich and detect phosphopeptides from complex digests.
• Measure single-field CCS values to compare phosphorylated versus nonphosphorylated peptide conformations.
• Resolve positional isomers and quantify structural differences arising from phosphorylation number and site.

Methodology and Instrumentation

This workflow employs:

• Automated sample preparation: Denaturation, reduction, alkylation, tryptic digestion, and phosphopeptide enrichment on Fe(III)-NTA cartridges using an automated liquid handler.
• Nanoflow LC: Agilent Infinity II 1290 binary pump delivering trap and analytical columns at low-nL/minute flow rates with a 65-minute gradient.
• NanoESI IMS-Q-TOF: Agilent 6560 ion mobility LC/Q-TOF with an ~80 cm drift tube, dual ion funnels, and alternating frames acquisition enabling concurrent MS and All-Ions MS/MS.
• Data analysis: Agilent MassHunter Qualitative and IM-MS Browser software for feature extraction, CCS calibration, averaged MS/MS spectra, and sequence confirmation.

Main Results and Discussion

Key findings include:

• Phosphopeptides exhibit consistently smaller CCS values compared to nonphosphorylated peptides of similar m/z, reflecting compaction upon addition of phosphate groups.
• Single-field CCS measurements, calibrated with standard tune mix ions, revealed distinct drift time distributions for peptides differing only in phosphorylation site location or number.
• Ion mobility separation resolved conformational isomers of doubly and triply phosphorylated peptides that coelute by LC alone. For example, the phosphopeptide VNELSKDIGSESTEDQAMEDIK displayed two drift peaks corresponding to alternative phosphorylation sites at positions 41, 46, or 48.
• Comparative CCS plots of peptide pairs with identical phosphorylation patterns but swapped residue order (e.g., RSpYpSRSR vs. RYpSpSRSR) demonstrated sequence-dependent folding differences detectable only via IMS.
• Alternating frames acquisition allowed simultaneous collection of CCS and high-energy fragmentation data. MS/MS fragments co-migrated with precursor drift times, supporting confident site assignment and near-complete sequence coverage for model phosphopeptides from commercial PhosphoMix standards.

Benefits and Practical Applications

This integrated IMS-MS approach offers:

• Enhanced phosphoproteome coverage by combining automated enrichment with high-resolution separation in four dimensions (m/z, retention time, drift time, intensity).
• Accurate CCS measurements to distinguish structural isomers and study phosphorylation-induced conformational changes.
• Efficient alternation between MS and All-Ions MS/MS, capturing fragmentation and mobility data in a single run without quadrupole isolation.
• Improved site localization confidence for low‐abundance or difficult‐to-fragment phosphopeptides.
• Potential extension to other post-translational modifications and complex proteomics samples in research and quality control environments.

Future Trends and Applications

Advances likely to expand IMS-MS in phosphoproteomics include:

• Higher‐throughput workflows integrating fast microflow IMS and parallel enrichment strategies for large cohort studies.
• Machine learning models trained on CCS libraries to predict and annotate novel phosphorylation sites and isomers.
• Integration with top-down and middle-down proteomics to study intact protein phosphorylation patterns in structural biology.
• Application of trapped ion mobility (TIMS) and structures for lossless ion manipulation (SLIM) devices offering greater resolution and sensitivity.
• Combined IMS and hydrogen-deuterium exchange measurements to correlate phosphorylation with dynamic conformational changes.

Conclusion

An automated IMS-MS workflow effectively enriches, separates, and characterizes site-specific phosphopeptides. Single-field CCS measurements reveal phosphorylation-dependent compaction and distinguish positional isomers and sequence variants not resolved by LC–MS alone. The ability to obtain simultaneous drift, m/z, and fragmentation data provides a powerful platform for in‐depth phosphoproteomic analysis, with broad applications in biochemical research, biomarker discovery, and industrial proteomics.

References

• Russell J., Murphy S. Agilent AssayMAP Bravo Technology Enables Reproducible Automated Phosphopeptide Enrichment. Agilent Technologies Application Note 5991-6073EN (2016).
• Wu L., Miller C.A. The Agilent Nanoadapter for Discovery Proteomics Using Nanoflow LC/MS. Application Note 5991-8174EN (2017).
• Mason E.A., McDaniel E.W. Transport Properties of Ions in Gases. John Wiley & Sons (1988).
• Stow S.M. et al. An Interlaboratory Evaluation of Drift Tube Ion Mobility–Mass Spectrometry Collision Cross Section Measurements. Anal. Chem. 89, 9048–9055 (2017).