Optimized Sample Preparation for Phospho-Enrichable Crosslinkers

Importance of the Topic

Cross-linking mass spectrometry enables mapping of protein–protein interactions in complex systems but is often limited by low yields of crosslinked peptides. Phospho-enrichable crosslinkers such as DSPP and TBDSPP combine high specificity enrichment with robust protocols, addressing the need for sensitive detection of low-abundance crosslinks in purified proteins and whole cells.

Study Objectives and Overview

This study aimed to optimize sample preparation workflows for DSPP (PhoX) and TBDSPP (tBu-PhoX) to improve crosslinked peptide identification. Key goals included refining crosslinking conditions, evaluating deprotection protocols, integrating phosphoenrichment, and adapting Thermo Scientific EasyPep chemistry for compatibility with enriched crosslinked samples.

Methodology and Instrumentation

The workflows encompassed:

• Crosslinking: DSPP and TBDSPP applied at defined molar ratios to BSA or HeLa cell lysates, with quenching and protein-level cleanup using PES filters or protein aggregation capture (PAC).
• Deprotection: Acid treatment with TFA (2–3% at 37 °C for 1 hour) to remove tert-butyl groups prior to enrichment.
• Phosphoenrichment: Fe-NTA magnetic agarose beads with optimized bead-to-sample ratios (1:10) and modified wash/elution buffers.
• Peptide cleanup and analysis: EasyPep chemistry with adjusted ionic strength, LC separation on Thermo Scientific UltiMate 3000 Nano LC columns, and detection on Orbitrap Eclipse, Q Exactive Plus/HF instruments.
• Data processing: Proteome Discoverer software with XlinkX node and a minimum confidence score of 40.

Key Results and Discussion

Optimized workflows delivered a 5–10× increase in crosslinked peptide identifications versus DSS or DSSO. MeCN solubilization and DMSO reconstitution maximized DSPP efficiency. Deprotection at the peptide level was superior to protein-level TFA treatments, which risked sample degradation. TBDSPP enabled in vivo crosslinking by permeating cell membranes, followed by efficient acid deprotection and phosphoenrichment. PAC outperformed acetone precipitation, improving cleanup and digestion without compromising recovery.

Benefits and Practical Applications

This optimized protocol:

• Enhances identification of low-abundance crosslinks in complex proteomes and intact cells.
• Reduces sample preparation time through streamlined EasyPep and PAC workflows.
• Enables structural proteomic studies in vivo with high confidence and throughput.

Future Trends and Potential Applications

Emerging directions include:

• Design of next-generation phospho-enrichable crosslinkers with broader reactivity and cleavable features.
• Integration with data-independent acquisition (DIA) and machine learning for deeper coverage.
• Single-cell crosslinking strategies to resolve cell-to-cell interaction heterogeneity.
• Adaptation to clinical samples and biopharmaceutical quality control.

Conclusion

The refined DSPP and TBDSPP workflows significantly improve crosslink detection in purified proteins and living cells. By combining optimized deprotection, phosphoenrichment, and EasyPep-compatible cleanup, the protocols deliver higher sensitivity and robustness, paving the way for advanced structural proteomics applications.

References

1. Steigenberger et al. Phox: an IMAC-enrichable Crosslinking Reagent. ACS Cent. Sci. 2019, 5, 1514–1522.
2. Pin Lian Jiang et al. A Membrane-Permeable and Immobilized Metal Affinity Chromatography (IMAC)-Enrichable Cross-Linking Reagent to Advance In Vivo Cross-Linking Mass Spectrometry. Angew. Chem. Int. Ed. 2021, e202113937.
3. Batth et al. Protein Aggregation Capture on Microparticles Enables Multipurpose Proteomics Sample Preparation. Mol. & Cell Proteomics 2019, 18, 1027–1035.