APPLICATION OF CYCLIC ION MOBILITY MASS SPECTROMETRY FOR HIGH PEAK CAPACITY SEPARATIONS OF NATIVE AND DEUTERATED PEPTIDE MIXTURES

Importance of the Topic

Integrating cyclic ion mobility with mass spectrometry addresses the need for high peak capacity separation in proteomics and structural studies. Rapid separations minimize sample consumption and reduce analysis times while preserving structural information in native and deuterated peptides.

Objectives and Study Overview

This study evaluated the performance of a SELECT SERIES cyclic ion mobility device coupled to a quadrupole time-of-flight mass spectrometer for rapid separation of enolase digest, model peptide P1 and myoglobin. Key aims were to compare infusion and fast liquid chromatography modes, explore multi-pass mobility resolution and assess deuterium retention under gas-phase conditions.

Methodology and Instrumentation

An enolase tryptic digest, a fully deuterated backbone model peptide and myoglobin were analyzed using rapid UPLC on a BEH C18 column and direct infusion. The cyclic IMS device provided 1 to 10 passes around a 98 cm path with nitrogen as mobility gas and helium at the entrance. Data were acquired with MassLynx and processed in DriftScope.

Main Results and Discussion

The cyclic IMS consistently achieved higher ion mobility resolution and peak capacity, fully disentangling overlapping isotope clusters in a congested m/z region. Infusion-only experiments detected comparable peptide coverage to rapid LC-MS. Controlled experiments on model peptide P1 revealed that backbone amide deuteriums remained stable across multiple passes while labile sidechain deuteriums exchanged. Multi-pass separation uncovered additional conformational families of myoglobin ions correlating with distinct deuterium uptake.

Benefits and Practical Applications

• Accelerated proteomic and HDX-MS workflows with minimal chromatography
• Enhanced resolution of complex mixtures under rapid analysis
• Structural insights from gas-phase conformations and deuterium labeling

Future Trends and Opportunities

Advances may include coupling cyclic IMS with advanced fragmentation techniques, automated multi-dimensional separations and expanded gas-phase chemistries to further probe protein dynamics. Integration into high throughput workflows will broaden applications in structural proteomics and QC analyses.

Conclusion

Cyclic IMS in single and multi-pass modes offers a powerful tool for rapid high peak capacity separations while preserving critical deuterium labeling. This approach enhances structural analysis of native and labeled peptides and proteins in accelerated proteomic workflows.