Bottom-up and Top-down Disulfide Bond Mapping of Beta-lactoglobulin on a Q-TOF with the Capability to Perform both CID and ECD

Importance of Disulfide Bond Mapping in Beta-lactoglobulin

Disulfide bonds play a crucial role in determining protein structure, stability, and function. Accurate mapping of these bonds is essential for characterizing protein folding, analyzing biotherapeutic quality, and understanding functional mechanisms in food and pharmaceutical applications.

Aims and Study Overview

The study aimed to demonstrate both bottom-up and top-down approaches for disulfide bond mapping of beta-lactoglobulin using a single quadrupole time-of-flight mass spectrometer retrofitted with an ExD cell. In particular, it compared collision-induced dissociation (CID) and electron capture dissociation (ECD) performance for intact protein and peptide analysis under nonreducing conditions.

Methodology and Instrumentation

The experimental workflow included two parallel strategies:

• Bottom-up analysis: Nonreduced beta-lactoglobulin was tryptically digested to yield disulfide-linked peptides. The digest was analyzed by LC-MS/MS on a 1290 Infinity II LC coupled to a 6545XT AdvanceBio LC/Q-TOF with dual Agilent Jet Stream ionization. Chromatographic separation used an AdvanceBio Peptide Plus column (2.1×150 mm, 2.7 μm) at 60 °C with a water/acetonitrile/formic acid gradient.
• Top-down analysis: Intact protein at 10 μM was directly infused at 0.75 mL/hr into the same Q-TOF instrument equipped with an ExD cell. Both CID and ECD fragmentations were performed without hardware modification, enabling comparison of backbone cleavage efficiency and disulfide bond cleavage.

A summary of key Q-TOF settings: dual ESI source, gas temperatures 250–300 °C, gas flows 11–13 L/min, capillary voltage 3500–5000 V, fragmentor 175 V, with reference mass at m/z 922.0098. The ExD cell enabled ECD and combined CID/ECD experiments.

Main Results and Discussion

• Top-down protein fragmentation at the 14+ charge state yielded 39% sequence coverage with CID alone, mainly at the N-terminus. ECD increased coverage to 48%, adding C-terminal fragments. Combining ECD with 10–20 V additional CID raised coverage further to 55%, demonstrating complementary fragmentation modes in a single MS/MS scan.
• Bottom-up peptide mapping confirmed the expected interpeptide disulfide between Cys106 and Cys119. ECD MS/MS provided clear masses for the individual peptide products after disulfide cleavage, whereas CID spectra lacked these definitive masses. Intrapeptide disulfide bonds were similarly characterized, with ECD offering higher confidence in linkage assignments.

Benefits and Practical Applications of the Method

• Unified platform: Ability to perform both CID and ECD on the same Q-TOF without hardware changes simplifies workflows and reduces instrument investment.
• Enhanced sequence coverage: ECD and combined CID/ECD deliver more complete fragmentation maps for intact proteins, improving structural characterization of disulfide-rich proteins.
• Higher confidence in disulfide assignments: ECD yields direct cleavage of disulfide bonds, producing clear product masses in bottom-up analyses for robust mapping of inter- and intrapeptide linkages.

Future Trends and Opportunities

Advances in high-resolution MS and novel fragmentation chemistries are expected to further enhance disulfide bond mapping. Integration of ultraviolet photodissociation or hybrid activation methods may complement ECD/CID. Automated data analysis pipelines leveraging machine learning could accelerate interpretation of complex fragmentation patterns, enabling routine disulfide profiling in biopharmaceutical development and quality control.

Conclusion

This work demonstrates effective bottom-up and top-down disulfide bond mapping of beta-lactoglobulin using a Q-TOF instrument capable of both CID and ECD. The combined approaches deliver detailed structural insights, improved sequence coverage, and confident disulfide localization without major hardware changes, offering a streamlined solution for protein characterization workflows.

Reference

Glaskin R, Hare M, Meeuwsen J, Knierman M, Beckman J. Bottom-up and top-down disulfide bond mapping of beta-lactoglobulin on a Q-TOF with the capability to perform both CID and ECD. ASMS 2022, Poster TP426.