Optimization of Crosslinked Peptide Analysis on an Orbitrap Fusion Lumos Mass Spectrometer

Significance of the Topic

Chemical crosslinking combined with mass spectrometry enables direct mapping of protein–protein interaction sites and structural interfaces in complex samples. Optimizing crosslinker chemistries and acquisition strategies improves identification rates, deepens structural insights, and advances applications in proteomics, structural biology, and quality control workflows.

Objectives and Overview

This study compared traditional non-cleavable crosslinkers (BS3, DSS) with MS-cleavable reagents (DSSO, BuUrBu) for labeling bovine serum albumin (BSA) and Escherichia coli lysates. It assessed labeling efficiency, peptide identification yields, and the impact of fragmentation methods (CID, HCD, ETD, EThcD) and tandem MS levels (MS2 vs. MS2–MS3) on crosslinked peptide detection.

Methodology

• Sample Preparation: Crosslinking of 2 mg/mL BSA in 50 mM HEPES pH 8 with varying molar excess (20×–500×) of each crosslinker; quenching with Tris buffer; reduction, alkylation, and tryptic digestion. E. coli lysates were treated similarly at 20× excess.
• Chromatography and Mass Spectrometry: Peptide fractionation via SCX (HyperSep Retain CX); RP-HPLC separation on an EASY-Spray column; analysis on Orbitrap Fusion Lumos and Q Exactive HF instruments. Acquisition parameters varied for MS2 CID/HCD, ETD, EThcD, and MS3 stages.
• Data Analysis: Proteome Discoverer 2.2 with XlinkX node and SEQUEST HT searches against BSA or E. coli databases at 1% FDR. MS2–MS3 linear-peptide search option employed for cleavable crosslinkers.

Instrumental Setup

• Orbitrap Fusion Lumos Tribrid mass spectrometer
• Q Exactive HF mass spectrometer
• Dionex UltiMate 3000 RP-HPLC system with EASY-Spray column
• HyperSep Retain CX strong cation exchange cartridges

Main Results and Discussion

Cleavable crosslinkers showed slightly lower BSA labeling efficiency than non-cleavable reagents, likely due to differences in linker properties. In BSA experiments:

• DSSO and BuUrBu identified >40 intercrosslinked peptides using MS2–MS3 workflows versus <20 with MS2 CID.
• MS2–MS3 combined with EThcD yielded the highest identification rates for cleavable reagents.
• Non-cleavable BS3 and DSS performed robustly across CID, HCD, and EThcD MS2 methods.
• Optimizing EThcD energy settings altered fragment intensity and unique peptide identifications.

In E. coli lysate analyses, the MS2–MS3 plus EThcD approach provided the greatest number of crosslinked peptide identifications compared to other acquisition schemes.

Benefits and Practical Applications

Enhanced crosslinking workflows support:

• Comprehensive mapping of protein assemblies in complex biological samples.
• Improved structural modeling of protein interfaces.
• Quality assurance in biopharmaceutical development.
• High-throughput proteome-wide interaction profiling.

Future Trends and Potential Applications

Ongoing developments may include novel cleavable linkers with improved solubility and reactivity, advanced fragmentation strategies, automated data analysis pipelines, integration with ion mobility, and deeper coverage in whole-cell and native complex studies.

Conclusion

MS-cleavable crosslinkers combined with optimized MS2–MS3 and EThcD workflows significantly enhance crosslinked peptide identification compared to traditional methods. Tailoring acquisition parameters and leveraging dedicated software nodes yields deeper insights into protein–protein interactions in both simple and complex samples.

References

• Kao A 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 MQ 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.