Rapid Antibody Structural Characterization and Quantification via Microdroplet Trypsin Digestion

Microdroplet digestion of proteins, including monoclonal antibodies (mAbs), has gained increasing attention for its potential in rapid protein structural characterization. In this study, we developed a fast antibody characterization and quantitation method using online microdroplet trypsin digestion. This automated approach involves ultrafast digestion in <1 ms (>90% digestion efficiency) and subsequent MS and MS/MS for peptide mapping and pinpointing peptide modifications. For instance, by MS/MS analysis, the N³⁸⁷ residue rather than N³⁹² and N³⁹³ of the "PENNYK" peptide, GFYPSDIAVEWESN³⁸⁷GQPEN³⁹²N³⁹³YK generated from NIST mAb microdroplet digestion, was found to undergo major deamidation due to its proximity to the G³⁸⁸ residue (upon incubation with a pH 8 Tris buffer for 5 days, ca. 32% of the N³⁸⁷ residue was deamidated). To further demonstrate our method's applications, methionine oxidation, another important type of post-translational modification (PTM) of mAbs, was successfully quantified by spiking the mAb sample with standard peptides for microdroplet digestion. Such an absolute quantitation approach showed a better accuracy for measuring the methionine oxidation level, in comparison to traditional relative quantitation methods, which simply compare ion intensities of oxidized and intact peptides to calculate the oxidation level (the relative quantitation is problematic as the oxidized and intact peptides have different ionization efficiencies). Similarly, by spiking the antibody with a heavy isotope-labeled antibody, our microdroplet digestion method allowed quick absolute antibody quantification, demonstrating good linearity (R² = 0.99), sensitivity (LOD: 1.2 ng), and accuracy (0.6-10% quantification error in comparison to the theoretical amount of injected antibody).