ACQUISITION MODE CHARACTERIZATION FOR THE QUANTITATIVE AND QUALITATIVE ANALYSIS OF CROSS-LINKED PEPTIDES BY TARGETED AND UNTARGETED LC-IM-MS

Significance of the Topic

Cross-linking mass spectrometry (XLMS) is a powerful technique for probing protein structures and interactions that are challenging to study by conventional high-resolution methods. Quantitative XLMS enhances our understanding of protein assemblies by providing both abundance and spatial information.

Objectives and Study Overview

This study aims to evaluate and compare various LC–ion mobility–MS acquisition modes for quantitative and qualitative analysis of cross-linked peptides. The authors assess targeted (PRM-style) and untargeted (DIA-style) methods, explore isotope correction strategies, and demonstrate how collisional cross section data and ETD fragmentation can enrich sequence information.

Methodology and Instrumentation

The workflow includes cross-linked bovine serum albumin (BSA) spiked into a complex four-protein digest matrix and cannabinoid metabolites in urine. Key instrumentation details are:

• AQUITY I-Class UHPLC with HSS T3 column (2.1×100 mm, 1.8 μm) and reversed-phase gradient at 0.4 mL/min.
• Waters M-Class nano-LC with BEH C18 column (0.3×100 mm, 1.7 μm) at 5 μL/min.
• Synapt G2-Si Q-ToF MS equipped with traveling-wave ion mobility (IM), SONAR scanning quadrupole DIA, and ETD using 4-nitrotoluene reagent.
• Acquisition modes compared: MS, HDMS, DDA, HDDDA, TofMRM, TofMRM-EDC, HDMRM, MSE, HDMSE, SONAR.
• Data processing: Skyline for peak detection, custom Python scripts, and MassLynx for qualitative interpretation.

Main Results and Discussion

The study highlights that IM-enabled DIA and PRM methods deliver superior dynamic range and sensitivity. Key findings include:

• IMS separation reduces chemical noise and enhances duty cycle, improving signal-to-noise by up to 10-fold.
• Isotopic correction based on unsaturated isotope peaks extends dynamic range by ~35% for IM-assisted methods.
• DIA (MSE, HDMSE, SONAR) and PRM (TofMRM, HDMRM) show low limits of detection (LLOD) and broad linear ranges for both metabolites and peptides.
• ETD fragmentation enables near-complete sequence coverage of cross­linked peptides, facilitating confident identification.
• A label-free Hi(n) quantitation approach estimates cross-link stoichiometry, revealing that cross-linked BSA peptides are present at ~10-fold lower molar abundance than linear peptides.

Benefits and Practical Applications

This comparative evaluation informs analysts on optimal acquisition strategies for XLMS workflows. Practical advantages include:

• Choice of DIA or PRM modes for high-throughput quantitation of cross-linked peptides.
• IMS integration to improve selectivity in complex biological samples.
• Isotope correction tools to maximize dynamic range in quantitative studies.
• ETD-based sequence annotation for structural validation of cross-links.

Future Trends and Opportunities

Advancements likely to drive the field include:

• Integration of advanced IM separations for multi­dimensional profiling of protein complexes.
• Development of standardized label-free quantitation software tailored to XLMS.
• Application of machine learning to predict cross-link behavior and optimize acquisition parameters.
• Expansion to in vivo cross-linking studies for mapping dynamic protein interactions.

Conclusion

The study systematically characterizes multiple LC–IM–MS acquisition strategies, demonstrating that IM-enabled DIA and PRM yield the best quantitative performance for cross-linked peptides. Combined with isotopic correction and ETD fragmentation, this approach facilitates sensitive, accurate, and comprehensive XLMS analyses.

References

• James et al. Optimization Workflow for the Analysis of Cross-Linked Peptides Using a Quadrupole Time-of-Flight Mass Spectrometer. Anal Chem. 2019;91(3):1808-1814.
• Lermyte et al. ETD Allows Surface Mapping of a 150 kDa Noncovalent Complex on a Commercial Q-TWIMS-TOF Instrument. J Am Soc Mass Spectrom. 2014;25(3):343-350.
• Helm et al. Ion Mobility Tandem MS Enhances Bottom-Up Proteomics. Mol Cell Proteomics. 2014;13(12):3709-3715.
• Daly et al. Qualitative and Quantitative Characterization of Plasma Proteins with Traveling Wave IM. Anal Chem. 2014;86(4):1972-1979.
• Silva et al. Absolute Quantification of Proteins by LC-MSE. Mol Cell Proteomics. 2006;5(1):144-156.
• Switzar et al. In-Depth Characterization of Protein Disulfide Bonds by Online LC-Electrochemistry-MS. J Am Soc Mass Spectrom. 2016;27(1):50-58.