Development and Application of SLIM-based Mobility-Aligned Fragmentation for Protein Analysis

Significance of the Topic

The integration of high-resolution ion mobility (HRIM) with mass spectrometry enhances separation and structural insight in proteomics.

Mobility-aligned fragmentation (MAF) exploits SLIM technology to align precursor and fragment ions by their drift times, delivering precise peptide identification without traditional quadrupole isolation.

Study Objectives and Overview

This work introduces MAF using a 13 m SLIM module coupled to a Q-TOF instrument, aiming to establish a robust data-independent fragmentation workflow.

A bovine serum albumin (BSA) tryptic digest served as a model system to validate LC-IM-MS/MS performance under varying gradient lengths and direct infusion conditions.

Methodology

A standardized BSA digest was analyzed by reverse-phase LC (30 min and 90 min gradients) and by direct infusion.

Ion mobility separation in the SLIM device was combined with low (0 V) and high (30 V) collision energies to generate precursor and product ion spectra.

Frames acquired at different energies were stitched based on arrival time distributions (ATDs) and processed in Skyline for peptide identification.

Instrumentation

• 13 m SLIM HRIM device (MOBIETM, MOBILion Systems)
• 6546 Q-TOF mass spectrometer (Agilent Technologies)
• 1290 Infinity II LC system (Agilent Technologies)
• HRIM Data Processor (HRIM-DP) and PNNL Preprocessor v3.0
• Skyline software with IM-MS Browser

Main Results and Discussion

MAF generated arrival-time aligned MS/MS spectra, with precursor and fragment ions coalescing in the drift time domain.

Sequence coverage of BSA peptides was highest in 90 min LC runs, followed by 30 min gradients; direct infusion confirmed proof of concept.

All MAF datasets were compatible with Skyline, enabling semi-automated analysis comparable to conventional DDA/DIA approaches.

Benefits and Practical Applications

• Eliminates quadrupole-based precursor isolation
• Enables rapid, data-independent MS/MS acquisition
• Fully integrates with established proteomics software for streamlined workflows
• Suitable for high-throughput QA/QC, industrial analytics, and academic research

Future Trends and Applications

Further coupling of MAF with advanced chromatographic and automation platforms will increase throughput and reproducibility.

Application to complex proteomes, post-translational modification analysis, and real-time AI-driven data processing will expand analytical capabilities.

Scaling SLIM-based fragmentation to larger biomolecules and multi-omic studies represents a key future direction.

Conclusion

Mobility-aligned fragmentation via SLIM technology offers a robust, quadrupole-free approach for peptide fragmentation and identification, demonstrating high sequence coverage and seamless software compatibility.

References

• Scientific Data. 2020;7(1):389.
• Nature Methods. 2014;11(2):167-170.
• Journal of Proteome Research. 2015;14(12):5378-5387.
• Analytical Chemistry. 2018;90(15):9529-9537.