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Updated: Jan 11, 2026
09:10
A Fast and Quantitative Method for Post-translational Modification and Variant Enabled Mapping of Peptides to Genomes
Published on: May 22, 2018
9.9K
Artifactual Sequence Variant Induced by Nonspecific Cleavage-Linked Transpeptidation in Peptide Mapping
Gangling Xu1,2, Gang Wu1,2, Chunyu Liu1,2
1Key Laboratory of the Ministry of Health for Research on Quality and Standardization of Biotech, National Institutes for Food and Drug Control, Beijing 102629, China.
Analytical Chemistry
|November 14, 2025
Summary
Analytical artifacts in therapeutic antibody development can be identified. This study reveals a novel artifactual sequence variant formation mechanism during peptide mapping, crucial for accurate cell line development and process optimization.
Area of Science:
- Biochemistry
- Analytical Chemistry
- Biotechnology
Background:
- Accurate identification of sequence variants is critical for therapeutic antibody safety and efficacy.
- Protein-level sequence variant analysis commonly uses LC-MS/MS peptide mapping.
- Mismatched mass spectral assignments and sample preparation artifacts can lead to incorrect variant identification.
Purpose of the Study:
- To report and elucidate the formation mechanism of a specific type of artifactual sequence variant in therapeutic antibodies.
- To differentiate artifactual variants from authentic sequence variants during data processing.
Main Methods:
- Liquid chromatography-tandem mass spectrometry (LC-MS/MS) peptide mapping.
- Analysis of antibody Fc region variants.
- Investigation of proteolytic digestion steps and nonspecific cleavage.
Main Results:
- A novel artifactual sequence variant (C425S → C425R) was identified in the antibody Fc region.
- The artifact arises from amino acid transfer to the C-terminus of nonspecifically cleaved peptides during digestion.
- Higher cleavage efficiency at carbamidomethyl-C425S compared to carbamidomethyl-C425R facilitates byproduct accumulation.
Conclusions:
- Artifactual sequence variants are generated through nonspecific cleavage-associated transpeptidation.
- These artifacts can be identified during data processing, improving the accuracy of therapeutic antibody analysis.
- Understanding these artifacts is essential for reliable cell line development and process optimization.
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