Highlights
- Simple and efficient workflow for studying post-translational modifications (PTMs) of monoclonal antibodies
- High-resolution LCMS-9030 Q-TOF combined with Byos software enables accurate quantification of relative abundance at amino acid sites
- Rigorous peptide mapping data review logic
Introduction
Antibody therapeutics hold enormous growth potential in the biopharmaceutical industry. Monoclonal antibodies (mAbs) are large protein molecules consisting of two identical light and heavy chains connected by disulfide bonds; functionally, they are divided into the antigen-binding fragment (Fab) and the crystallizable fragment (Fc). mAbs can be identified through unique complementarity-determining regions (CDRs) on the heavy and light chains. Throughout the drug development lifecycle, liquid chromatography-mass spectrometry (LC-MS) is widely used for antibody structural characterization, with peptide mapping providing rich primary structural information.
Structure of monoclonal antibody
In this study, the NIST reference protein was used for peptide mapping. Trypsin enzyme was used to digest the mAbs into smaller peptide fragments. The digested peptides were then separated using liquid chromatography and their masses were detected using Shimadzu LCMS-9030 QTOF Data Dependent Acquisition (DDA) mode. Data processing was performed using Protein Metrics software to quantify the amino acid site relative abundance.
Q-TOF mass spectrometer LCMS-9030
Experimental
Reagents and Chemicals
NISTmAbs Humanized IgG1k monoclonal antibody reference material 8671 was obtained from Merck Pte Ltd. Sequencing Grade Modified Trypsin (V511) was obtained Promega. S-TrapTM mini spin columns was obtained from Protifi. TORAST-H Bio Vial (370-04350-00) was obtained from Shimadzu.
Acetonitrile (LCMS grade) was obtained from commercial suppliers. Formic acid (>99%) of LCMS grade was used as additive in the mobile phase prepared from Milli-Q water.
Sample Preparation
100 µg of NISTmAb was first reduced using 1 M dithiothreitol (DTT) by heating it at 95ºC for 10 mins. Then, it was alkylated using 100 mM iodoacetamide (IAM) by incubating it in the dark at room temperature for 30 mins. The reaction was quenched by adding 12% phosphoric acid. Next, the mAbs were transferred to the S-trap mini spin column and washed with 90% methanol in 100 mM triethylammonium bicarbonate (TEAB). The mAbs were then captured on the S-Trap mini spin column. Trypsin digestion was performed using a 1:20 (w/w) enzyme-to-protein ratio and incubating at 47ºC for 1 hr. The digested peptides were eluted using 50% acetonitrile: 50% water: 0.2% formic acid. Peptides were dried using refrigerated vacuum concentrate at 4ºC and reconstituted in 80µl of 0.1% formic acid in water in a low protein TORAST-H Bio vial.
Data Analysis
One of the key challenges in peptide mapping is evaluating the reliability of peptides reported by the software. Byos address this through rigorous review logic to ensure that every identified peptide is confident. Unmodified peptides identified by Byos are almost always reliable, so users need only focus on reviewing modified peptides.
The review logic for modified peptides covers three main criteria: retention time (the retention time of a modified peptide differs from its unmodified counterpart), MS1 (the observed monoisotopic peak matches the theoretical monoisotopic peak), and MS2 (the difference in characteristic fragment ions between the modified and unmodified peptides is consistent with the delta mass of the modification, or you can also check Score and Delta Mod. Score for confrimation). Only when all three criteria are satisfied is an identified peptide considered confident; if any one criterion is not met, the peptide is considered a false positive.
Score reflects the absolute quality of PSM. Byonic scores range from 0 to about 1000, with 300 a good score, 400 a very good score, and PSMs with scores over 500 almost sure to be correct.
Delta Mod. Score gives an indication of whether modifications are confidently localized; DeltaMod.Score over 10.0 means that there is a high likelihood that all modification placements are correct.
Byos also supports Mod Validator, which allows a user to assess the validity of a PTM automatically. When this feature is activated, the algorithm will iterate row by row and assess whether the modification meets or fails certain thresholds, and gives Warning, Critical or Clear lables in the Comment field.
For more information about Mod Validator, you can reach out to support@proteinmetrics.com.
True Peptides
An intuitive user interface allows users to simultaneously view modified and unmodified peptides. Selecting any modified peptide automatically updates the corresponding XIC, MS1, and MS2 views. Each panel is split into upper and lower stacked plots — the upper showing the modified peptide and the lower showing the unmodified peptide.
As peptide molecular weight increases, the relative abundance of the monoisotopic peak decreases and may sometimes be undetectable. Clicking "Enable MS1 Summed" allows MS1 spectra to be summed, improving MS1 sensitivity.
If there is a strong MS1 interference peak at a certain time point in the raw data, one or more peaks may still be visible before the blue dot even with "Enable MS1 Summed" activated. In this case, clicking "Enable MS Scan Extraction" allows viewing the MS1 spectrum at each individual time point. If the peak before the blue dot appears only at a specific time point, it can be confirmed as an interference peak, and the software-identified monoisotopic peak is correct. If the peak before the blue dot appears consistently across time points, it can be confirmed as the observed monoisotopic peak, indicating a mismatch between the theoretical and observed values — making it a false positive peptide.
False Positive Peptides
PTM alters peptide hydrophobicity and generally result in a different retention time from the unmodified peptide.
During peptide identification, the software may select an incorrect monoisotopic peak, giving false positive data.
Ambiguous Peptides
When retention time and MS1 are both correct but MS2 information is insufficient, additional supporting information is required for cross-validation. The example shown below involves a dehydration modification that could occur at Y3 or Y7, and the DeltaMod. Score is 0.0 < 10.0. Due to the lack of characteristic fragment ions, the software may randomly assign a site, resulting in lower confidence for this peptide, (but it cannot be classified as a false positive).
Results
100% sequence coverage
PTM modification percentages
Conclusion
During drug development, it is critical to closely monitor changes in the relative abundance of PTMs, such as methionine oxidation or asparagine deamidation, as these can affect the efficacy, stability, and immunogenicity of monoclonal antibodies.
The Shimadzu LCMS-9030 Q-TOF mass spectrometer, combined with Protein Metrics software, provides high mass resolution to accurately identify, separate, and quantify the modified peptides with small mass shifts. Peptide mapping analysis offers deep insights into antibody characteristics, supporting drug development from early to late stages.
Acknowledgements
With thanks to Shannie Tay and Max Kosok of Shimadzu Asia Pacific for providing the raw data and analysis results.
References
Development of an LC-MS/MS peptide mapping protocol for the NISTmAb, Trina Mouchahoir, John E. Schiel, Analytical and Bioanalytical Chemistry (2018) 410:2111–2126
Microheterogeneity of Recombinant Antibodies: Analytics and Functional Impact, Beate Beyer, Manfred Schuster, Alois Jungbauer, and Nico Lingg, Biotechnol. J. 2017, 1700476