Relative Quantification of TMT-labeled, cross-linked proteins using XlinkX node in Proteome Discoverer

Importance of the topic

The structural organization and dynamic interactions of protein complexes underpin many cellular processes. Quantitative cross-linking mass spectrometry (QXL-MS) bridges structural biology and proteomics by capturing residue‐level proximity information in a multiplexed fashion. Implementing robust, high‐throughput workflows for isobaric‐tagged crosslinked peptides significantly enhances our capability to monitor conformational changes and protein–protein interactions under varying conditions.

Objectives and study overview

This work presents a complete analytical pipeline in Thermo Scientific™ Proteome Discoverer™ 2.3 for simultaneous identification and multiplexed quantification of DSSO‐crosslinked peptides labeled with tandem mass tags (TMT). The study demonstrates method validation using a Cytochrome C digest standard and applies the workflow to probe conformational shifts in the rabbit 20S proteasome upon activation by sodium dodecyl sulfate (SDS).

Methodology and instrumentation

Sample preparation involved cross-linking model proteins (Cytochrome C and 20S proteasome) with DSSO at defined protein:crosslinker ratios, followed by enzymatic digestion, TMT labeling, and prefractionation via strong cation exchange and size‐exclusion chromatography. Peptides were separated by reversed‐phase UHPLC and analyzed on an Orbitrap Fusion Lumos tribrid mass spectrometer using a suite of hybrid acquisition methods (ID‐MS3, SPS‐MS3, combined MS3–SPS) to capture both identification and reporter‐ion quantification. Data processing used Proteome Discoverer 2.3 with the XlinkX 2.0 node for crosslinked spectrum matches (CSMs) and corrected reporter abundances for isotopic impurities.

Main results and discussion

Among tested acquisition schemes, the ID‐SPS‐MS3 approach delivered the best balance of identification depth and quantitative accuracy, yielding over 100 unique intra- and interprotein crosslinks. Validation with TMT2‐plex Cytochrome C mixtures confirmed linear quantification over a 1:1, 1:5, and 1:10 ratio range. Application to the rabbit 20S proteasome revealed SDS‐induced conformational rearrangements in the outer alpha ring, notably changes in crosslink distances between Lys254 and Lys199. Quantitative comparisons of monolinked versus unmodified peptides showed consistent abundances, underscoring method precision.

Benefits and practical applications

• Enables simultaneous mapping and quantification of crosslinked networks within protein complexes.
• Supports comparative structural analysis under varying biochemical conditions.
• Integrates seamlessly into existing proteomics pipelines using widely adopted software.

Future trends and applications

Looking ahead, extending multiplex capacity with higher‐plex TMT reagents, integrating machine learning for crosslink assignment, and combining QXL-MS data with cryo‐EM or molecular dynamics simulations will drive deeper insights into proteome‐scale structural dynamics. Automation of sample preparation and real‐time data processing will further increase throughput and reproducibility.

Conclusion

The described Proteome Discoverer 2.3 workflow with XlinkX 2.0 offers a validated, high‐precision strategy for relative quantification of TMT‐labeled crosslinked peptides. Its robust performance in model proteins and the rabbit 20S proteasome demonstrates its value for probing conformational changes and protein interactions at residue resolution.

Instrumentation used

• Thermo Scientific™ UltiMate™ 3000 UHPLC system with EASY‐Spray™ column
• Orbitrap Fusion Lumos Tribrid Mass Spectrometer
• Proteome Discoverer™ 2.3 software with XlinkX 2.0 node

References

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3. Shibatani T., Ward W.F. Sodium dodecyl sulfate (SDS) activation of the 20S proteasome in rat liver. Arch. Biochem. Biophys. 1995;321(1):160–166.