INVESTIGATING PRIMARY AND HIGHER-ORDER PROTEIN STRUCTURE ANALYSIS USING A NOVEL CYCLIC ION MOBILITY ENABLED QTOF PLATFORM

Význam tématu

High-performance separations are critical for characterizing complex biotherapeutic and protein samples. Traditional liquid chromatography (LC) and mass spectrometry (MS) methods can struggle to differentiate isomeric or conformational variants. Ion mobility spectrometry (IMS) adds an orthogonal gas-phase separation based on collisional cross section and charge, enhancing resolution of co-eluting, isobaric, or structurally distinct species. A novel cyclic IMS-enabled quadrupole time-of-flight (QTOF) platform expands this capability with selectable high IMS resolution and IMS-MSn workflows.

Cíle a přehled studie / článku

The study introduces and evaluates the Waters SELECT SERIES Cyclic IMS Q-cIMS-oaTOF instrument. Key objectives include:

• Demonstrate scalable IMS resolution via multiple cyclic passes.
• Assess IMS-IMS and IMSn for targeted peptide and protein structural analysis.
• Apply the system to distinguish peptide isomers, resolve protein conformers, and study unfolding pathways.

Použitá metodika a instrumentace

This work employed the Q-cIMS-oaTOF MS featuring:

• A cyclic IMS cell positioned between the quadrupole and TOF detector.
• Selectable IMS cycle count to tune resolution (up to ~100 cycles, ~800 resolution units).
• IMS-IMS and IMSn modes with ion selection and fragmentation before, within, or after IMS separation.
• Optional high-resolution MS detection (60,000 to 100,000 resolving power).

Hlavní výsledky a diskuse

1. Peptide Isomer Resolution

• A 1:1 mixture of Asp and isoAsp tryptic peptide forms from trastuzumab heavy chain was baseline separated by either UPLC or a single IMS pass, demonstrating reduced chromatographic load.
• Fragment ions (b4, b10) retained IMS separability even after 10 IMS cycles, confirming structural specificity of peptide fragments.

2. Enhanced Peak Capacity

• Three co-eluting yeast enolase peptides resolved to a 10 % valley using three IMS cycles, illustrating effective peak capacity improvement in peptide mapping.

3. Native Protein Conformers and Unfolding Pathways

• Native infusion of cytochrome c (7+ charge) revealed distinct conformers under low energy.
• Selection and activation of the ‘alpha’ conformer followed by reanalysis unveiled additional beta, gamma, delta, epsilon, and zeta forms, mapping gas-phase unfolding trajectories.

4. Monoclonal Antibody Differentiation

• Co-infusion of IgG1 and IgG2 yielded resolved MS spectra; one IMS cycle identified a single IgG1 conformer and two major IgG2 forms, demonstrating capability in differentiating closely related therapeutic antibodies.

Přínosy a praktické využití metody

The cyclic IMS QTOF platform offers:

• Adjustable IMS resolution to tackle complex peptide and protein mixtures.
• IMS-IMS and IMSn workflows for targeted structural identification and fragmentation mapping.
• Enhanced separation of isomeric, conformational, and co-eluting species without extensive chromatography.

Budoucí trendy a možnosti využití

Anticipated developments include:

• Integration with hydrogen-deuterium exchange (HDX-MS) to probe protein dynamics without deuterium scrambling.
• Automated IMS-based proteoform screening in biopharmaceutical quality control.
• Expanded IMS-MSn libraries for structural elucidation of post-translational modifications.
• Combination with microfluidic LC front-ends for high-throughput native MS analyses.

Závěr

The SELECT SERIES Cyclic IMS Q-cIMS-oaTOF platform demonstrates unprecedented control over ion mobility resolution and multi-dimensional separation workflows. By enabling cyclical IMS passes and IMSn capabilities, it addresses limitations of traditional LC-MS in resolving isomeric, conformational, and co-eluting biomolecules, paving the way for deeper insights in protein structure, biotherapeutic characterization, and dynamic unfolding studies.

Reference

1. Berger SJ, Cooper-Shepherd DA, Palmer M, Martin LB, Shion H, Langridge JI. Investigating primary and higher-order protein structure analysis using a novel cyclic ion mobility enabled Q-cIMS-oaTOF platform. Waters Corporation; 2019.
2. Waters Corporation. SELECT SERIES Cyclic IMS QTOF MS poster. 2019.