A Qualitative and Quantitative Ion Mobility MS-Enabled, Data-Independent SILAC Workflow

Importance of the Topic

Stable isotope labeling by amino acids in cell culture (SILAC) is a cornerstone technique for accurate relative quantitation in proteomics. Integrating ion mobility with data-independent acquisition (HDMSE) enhances separation, reduces spectral interferences and improves quantitation precision in complex biological samples.

Objectives and Study Overview

This study demonstrates a workflow combining SILAC labeling with ion mobility-enabled data-independent LC-MS (HDMSE) on a SYNAPT G2-S instrument. The goal was to assess the accuracy, precision and practical applicability of quantitative proteomic analysis in a Jak2 V617F mutant murine pro-B cell line model.

Methodology

Murine pro-B cells harboring the oncogenic Jak2 V617F mutation were cultured in media containing either natural (“light”) lysine or 13C6-labeled (“heavy”) lysine. After several divisions, cells were lysed, protein concentrations measured and equal amounts of light and heavy lysates mixed (1:1). Proteins were digested with trypsin, and peptide mixtures were separated by reversed-phase nanoLC.

Used Instrumentation

• nanoACQUITY UPLC system with Symmetry C18 trap and BEH C18 analytical columns
• Waters SYNAPT G2-S Mass Spectrometer with T-Wave ion mobility
• RapiGest SF surfactant for enhanced digestion efficiency
• ProteinLynx Global SERVER software with Expression algorithm for identification and quantification

Main Results and Discussion

HDMSE acquisition alternated low- and high-energy scans, aligning precursor and fragment ions by retention time and drift time. Using modification reagent groups in the software, light/heavy peptide pairs were identified and quantified. Profilin-1 was quantified from 10 peptides with RMS mass errors of 3.2 ppm (precursor) and 4.2 ppm (product), yielding a log ratio of –0.92 ± 0.11. Overall protein ratio distribution showed a median of 0.47, mean of 0.48 and variance of 0.05 (ln scale). Approximately 80 % of quantified proteins fell within a 0.4–0.6 fold range without normalization.

Benefits and Practical Applications

• Enhanced quantitation precision through ion mobility separation
• Reduced co-fragmentation and improved identification rates
• Unbiased, simultaneous analysis of multiple labeled samples
• Seamless integration of identification and quantification workflows

Future Trends and Possibilities for Use

Future developments may include automated normalization routines, expansion to multi-channel SILAC, integration with advanced bioinformatics for pathway analysis and application to increasingly complex clinical and environmental proteomes.

Conclusion

The ion mobility-enabled HDMSE workflow on a SYNAPT G2-S provides a robust, precise and high-throughput platform for SILAC-based quantitative proteomics. It holds promise for routine applications in research, drug discovery and clinical biomarker validation.

References

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