Analysis of Modification Site of Chemically Modified Antibody Using MALDImini™-1 Compact MALDI Digital Ion Trap Mass Spectrometer

Significance of the topic

Antibody drug conjugates (ADC) combine the targeting ability of monoclonal antibodies with the potency of cytotoxic drugs to improve treatment selectivity and reduce systemic toxicity. Accurate characterization of the drug load and binding sites on the antibody is critical for product quality and therapeutic efficacy.

Objectives and study overview

This study creates a pseudo ADC by selectively modifying tryptophan residues on a standard monoclonal antibody with a low molecular weight fluorescent tag. The main goal is to demonstrate site specific mapping of the modification using a compact MALDI digital ion trap mass spectrometer.

Methodology

Tryptic digestion of both modified and unmodified antibodies was performed, followed by desalting with a C18 tip and spotting on a MALDI target. 2.5–dihydroxybenzoic acid was used as matrix. Mass spectra were acquired on a compact MALDI digital ion trap system and compared to detect modification specific ions. Target ions were subjected to MS/MS and MS3 for peptide backbone fragmentation and site localization. Peptide identities were confirmed by database search.

Instrumentation

• Compact MALDI digital ion trap mass spectrometer (MALDImini-1)
• ZipTip µC18 desalting tips
• 2.5-dihydroxybenzoic acid matrix

Main results and discussion

Comparison of mass spectra revealed four unique ions (m/z 2416.9, 2430.7, 2452.7, 2560.9) in the modified antibody. MS/MS and MS3 analyses localized the fluorescent tag to tryptophan-containing peptides from the heavy chain: sequence FNWYVDGVEVHNAK and sequence WSVLTVLHQDWLNGK. Mascot search confirmed these assignments. Observed mass shifts indicated elimination or substitution by CH2 or H/Na. Data suggest up to three modifications per antibody molecule, consistent with heavy and light chain stoichiometry.

Benefits and practical applications

The results illustrate that a benchtop MALDI digital ion trap instrument can provide high-resolution MSn data for site specific analysis of protein modifications. This approach enables rapid quality control and structural characterization of antibody drug conjugates and other bioconjugates in research and development environments.

Future trends and potential applications

Advances may include integration of liquid chromatography for peptide separation, higher multiplexing of modification sites, and expansion to other targeted conjugation chemistries. Further miniaturization and automation of compact MALDI platforms will increase throughput for biopharmaceutical analytics.

Conclusion

The compact MALDI digital ion trap mass spectrometer demonstrated efficient MSn capability for mapping chemical modifications on antibody peptides. This method supports detailed characterization of ADCs and contributes to improved quality control in antibody-based therapeutics development.

References

• Yohei Seki, Takashi Ishiyama, Daisuke Sasaki, Junpei Abe, Youhei Sohma, Kounosuke Oisaki, Motomu Kanai. Transition Metal-Free Tryptophan-Selective Bioconjugation of Proteins. Journal of the American Chemical Society. 2016;138(34):10798-10801.