Accurate Collision Cross Sections From Traveling Wave Ion Mobility Measurements in Native Mass Spectrometry – A Structural Investigation of Cas9 Ribonucleoprotein Complexes

Importance of the Topic

Traveling wave ion mobility spectrometry (TWIMS) paired with native mass spectrometry has become indispensable for probing the three-dimensional shapes of biomolecules. The collision cross section (CCS) derived from TWIMS serves as a quantitative indicator of ion conformation in the gas phase, informing on folding, complex assembly, and interactions. Accurate CCS measurements are particularly critical for understanding large protein complexes and ribonucleoproteins, such as Cas9–sgRNA assemblies that underpin CRISPR-based gene therapy applications.

Study Objectives and Overview

This study aimed to evaluate the IMScal software tool for calibrating TWIMS data on Waters™ instruments to yield precise CCS values. After verifying calibration quality against standard proteins, the team applied IMScal to measure CCS of recombinant Streptococcus pyogenes Cas9 and its complexes with a synthetic guide RNA (sgRNA). The goal was to demonstrate structural insights into Cas9 ribonucleoprotein formation using the SELECT SERIES™ Cyclic™ IMS mass spectrometer.

Methodology and Used Instrumentation

Sample Preparation:

• Calibrants: polyalanine oligomers and bovine serum albumin.
• Proteins: streptavidin, concanavalin A, Cas9 endonuclease.
• sgRNA targeted to human CA6 gene; Cas9 RNP formed by 1:2 protein:RNA incubation.

Instrumental Conditions:

• Mass spectrometer: SELECT SERIES™ Cyclic IMS.
• Ionization: static nanospray with borosilicate capillaries, positive mode, 1 kV capillary voltage.
• Ion mobility settings: single pass, cyclic wave height 20 V, wave velocity and waveform parameters defined in IMScal defaults.
• Acquisition m/z range: 50–16,000; trap collision energy 6 V; cone voltage 40–150 V.

Software and Calibration:

• Data processed with MassLynx™ v4.2 and Driftscope™ v3.0.
• IMScal GUI used a minimal calibrant set (small singly/doubly charged polyalanine plus BSA).
• Instrument geometry and wave parameters entered via default_settings.dat.

Main Results and Discussion

Calibration Quality Check:

• Streptavidin and concanavalin A CCS matched drift-tube references to within 0.3 %, confirming method accuracy.

Cas9 and RNP Analysis:

• Cas9 alone (27+ charge state) yielded CCS of 7893 ± 119 Ų, mass 162 kDa, single folded conformation.
• Free sgRNA exhibited CCS of 2280 ± 13 Ų at 8+ charge state, mass ~32.4 kDa.
• Cas9+1 sgRNA complex measured 8573 ± 170 Ų (194.5 kDa), suggesting sgRNA insertion into Cas9 cavity.
• Cas9+2 sgRNA species showed CCS 9891 ± 202 Ų (227 kDa), indicating external second RNA binding in absence of DNA.

These findings illustrate high-resolution separation and CCS determination of Cas9 complexes and reveal binding stoichiometry dynamics relevant for gene-editing research.

Benefits and Practical Applications

• Rapid, user-friendly calibration of TWIMS data for diverse analytes using IMScal.
• High accuracy CCS values (<0.3 % deviation) for proteins and complexes.
• Enhanced confidence in native MS structural studies, reducing ambiguity in conformational assignments.
• Applicability to QA/QC workflows in biotech and pharmaceutical development.

Future Trends and Opportunities

Advancements in TWIMS calibration and instrumentation will enable:

• Expanded CCS databases for non-canonical proteins, nucleic acids, and multifunctional assemblies.
• Integration of CCS data with in silico modeling and machine learning for predictive structure elucidation.
• Automation of calibration workflows and real-time data analysis in high-throughput screening.
• Application of native ion mobility MS in regulatory environments for gene therapy product characterization.

Conclusion

The combination of the IMScal calibration tool and the SELECT SERIES Cyclic IMS mass spectrometer delivers accurate, reliable CCS measurements for native biomolecules. Validated against standard proteins and applied to Cas9–sgRNA complexes, the workflow provides detailed conformational data crucial for structural biology and therapeutic development.

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