Proton transfer charge reduction (PTCR)

Importance of the Topic

Recombinant monoclonal antibodies have become cornerstone biotherapeutics in industry and clinical applications. Detailed primary sequence characterization of these ~150 kDa proteins and their ~25 kDa subunits is critical to ensure product safety, efficacy, and proper localization of post-translational modifications. Middle-down mass spectrometry bridges the gap between bottom-up and top-down approaches by enabling efficient subunit analysis with improved throughput and minimized sample handling artifacts.

Objectives and Study Overview

This work evaluates the benefits of coupling proton transfer charge reduction (PTCR) to electron transfer dissociation (ETD) for middle-down analysis of monoclonal antibody subunits. Key goals include: comparing standard ETD MS2 versus ETD MS2 followed by PTCR MS3 on an Orbitrap Eclipse Tribrid platform; assessing gains in sequence coverage for an IgG1 standard; and optimizing MS acquisition parameters for robust subunit characterization.

Methodology and Instrumentation

Sample preparation used the SILuLite SigmaMAb standard digested with IdeS protease, reduced with TCEP, and desalted via Zeba spin columns. Reverse-phase UHPLC was performed on a Vanquish Horizon system with a MAbPac RP column under denaturing conditions. Mass analysis employed an Orbitrap Eclipse Tribrid equipped with ETD and PTCR reagents in high-pressure octopole ion trap, using HMRn mode up to m/z 8000. Targeted MS2 and sequential PTCR MS3 experiments were conducted on selected precursor charge states with optimized reaction durations.

Main Results and Discussion

Implementing PTCR after ETD dramatically simplified product ion spectra by reducing fragment charge states and dispersing them across a wide m/z range. For the light chain subunit, sequence coverage increased from 46% (ETD MS2) to 64% (ETD MS2–PTCR MS3) within a single 25 min LC run. Complementary c/z ion pair identifications rose from 3 to 53, and large fragments (10–20 kDa) were readily matched with high mass accuracy. Comparable enhancements were observed for the Fd′ and Fc/2 subunits.

Benefits and Practical Applications

This combined ETD–PTCR strategy enables more comprehensive sequence mapping and confident localization of modifications in monoclonal antibody subunits without extending analysis time. It supports quality control and structural assessment in biopharmaceutical research, facilitating rapid and reliable characterization of antibodies and antibody-drug conjugates.

Future Trends and Opportunities

Integrating PTCR with additional activation methods such as CID, HCD, EThcD, or UVPD promises to further elevate sequence coverage. Ongoing advancements in high m/z detection, automated charge reduction, and data analysis pipelines will drive more in-depth intact and middle-down protein studies at higher throughput.

Conclusion

Proton transfer charge reduction coupled with ETD on an Orbitrap Eclipse Tribrid mass spectrometer effectively reduces spectral congestion, enhances detection of both small and large fragment ions, and significantly improves sequence coverage in middle-down antibody analysis within a single LC-MS workflow.

References

1. Srzentić K., et al. Interlaboratory Study for Characterizing Monoclonal Antibodies by Top-Down and Middle-Down Mass Spectrometry. J Am Soc Mass Spectrom. 2020;31:1783–1802.
2. Fornelli L., Ayoub D., Aizikov K., Beck A., Tsybin Y.O. Middle-Down Analysis of Monoclonal Antibodies with Electron Transfer Dissociation Orbitrap Fourier Transform Mass Spectrometry. Anal Chem. 2014;86:3005–3012.
3. Fornelli L., et al. Accurate Sequence Analysis of a Monoclonal Antibody by Top-Down and Middle-Down Orbitrap Mass Spectrometry Applying Multiple Ion Activation Techniques. Anal Chem. 2018;90:8421–8429.
4. Kline J.T., et al. Sequential Ion–Ion Reactions for Enhanced Gas-Phase Sequencing of Large Intact Proteins in a Tribrid Orbitrap Mass Spectrometer. J Am Soc Mass Spectrom. 2021;doi:10.1021/jasms.1c00062.