IMSC: Optimization of crosslinked peptide analysis on an Orbitrap Fusion Lumos mass spectrometer

Significance of the topic

Chemical crosslinking coupled to mass spectrometry is a cornerstone technique for mapping protein–protein interactions and distance constraints within protein assemblies. The advent of MS-cleavable crosslinkers and advanced fragmentation methods allows more confident peptide identification, deeper structural insights and applications ranging from purified complexes to whole‐cell lysates.

Objectives and study overview

This work systematically compares two traditional non-cleavable crosslinkers (DSS, BS3) with two MS-cleavable reagents (DSSO, BuUrBu) on a bovine serum albumin (BSA) standard and an Escherichia coli lysate. It evaluates crosslinking efficiency, peptide identification rates and the impact of fragmentation (CID, ETD, EThcD) and acquisition schemes (MS2, MS3) on a Thermo Scientific Orbitrap Fusion Lumos and a Q Exactive HF platform.

Methodology and instrumentation

Sample preparation and crosslinking

• BSA and E. coli lysates were crosslinked at 20× to 500× molar excess in 50 mM HEPES, pH 8.0, then quenched with Tris buffer.
• Proteins were reduced, alkylated, digested with trypsin, and peptide concentrations determined by BCA and colorimetric assays.
• Peptides were fractionated on a HyperSep Retain CX column using stepwise ammonium acetate elution and desalted by C18 SPE.

LC-MS/MS acquisition

• Separation by RP-HPLC on a Dionex UltiMate 3000 with EASY-Spray 50 cm × 75 µm column, 4–40 % ACN gradient at 300 nL/min.
• Analysis on Orbitrap Fusion Lumos with MS2 and MS3 acquisitions, combining CID, ETD and EThcD; Q Exactive HF used for complementary MS2 CID/HCD runs.
• Key MS parameters (resolution, AGC targets, max inject times, isolation widths and NCE) were optimized for each workflow.

Data analysis

• Proteome Discoverer 2.2 with XlinkX node and SEQUEST HT engine at 1 % FDR.
• Static modification: carbamidomethylation of cysteine; variable: methionine oxidation and lysine–crosslink adducts.
• MS2–MS3 workflows exploited MS3 scans for linearized peptide identification and improved crosslink filtering.

Key results and discussion

On the BSA standard, MS-cleavable crosslinkers (DSSO, BuUrBu) yielded over 40 unique inter-crosslinked peptide pairs using MS2-MS3/EThcD workflows, compared to fewer than 20 identifications with MS2 CID alone. Non-cleavable reagents (DSS, BS3) showed comparable efficiencies at high crosslinker excess but lagged behind in depth of identification. In the E. coli lysate, combining MS2–MS3 acquisition with EThcD delivered the highest number of crosslinked peptide identifications, outperforming pure MS2 CID or ETD methods. Variation of EThcD energy influenced fragment ion intensities and the spectrum of peptides detected.

Benefits and practical applications

• MS-cleavable reagents paired with MS3/EThcD strategies enhance identification confidence and proteome coverage.
• XlinkX integration in Proteome Discoverer streamlines data processing and spectra annotation.
• Optimized workflows support mapping of interactions in complex samples, facilitating structural modelling and systems biology studies.

Future trends and applications

Development of next-generation cleavable crosslinkers with tailored chemistries, higher-throughput MSn acquisition methods, and integration with quantitative XL-MS approaches will further expand proteome-wide interaction mapping. Advances in instrumentation speed, real-time search and AI-driven spectral interpretation promise deeper coverage and more rapid results in structural proteomics.

Conclusion

MS-cleavable crosslinkers (DSSO, BuUrBu) combined with MS2–MS3 acquisition and EThcD fragmentation on modern Orbitrap platforms significantly improve crosslinked peptide identification over traditional CID-only methods. The optimized workflows deliver robust coverage in both model proteins and complex cell lysates, supporting comprehensive mapping of protein interaction networks.

References

• Kao A. C. et al. Development of a novel cross-linking strategy for fast and accurate identification of cross-linked peptides of protein complexes. Mol. Cell Proteomics 2011, 10(1):M110.002212.
• Müller M. Q. et al. Cleavable cross-linker for protein structure analysis: reliable identification of cross-linking products by tandem MS. Anal. Chem. 2010, 82(16):6958–6968.
• Liu F. et al. Proteome-wide profiling of protein assemblies by cross-linking mass spectrometry. Nat. Methods 2015, 12(12):1179–1184.