Complementary Methods for de Novo Monoclonal Antibody Sequencing to Achieve Complete Sequence Coverage

Area of Science:

• Proteomics
• Analytical Chemistry
• Biochemistry

Background:

• Mass spectrometry is crucial for de novo protein sequencing.
• Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) is the predominant technique.
• Novel methods are needed for efficient and accurate antibody sequencing.

Purpose of the Study:

• To introduce and evaluate capillary electrophoresis tandem mass spectrometry (CE-MS/MS) for de novo sequencing of novel monoclonal antibodies.
• To compare the performance of CE-MS/MS against LC-MS/MS for antibody sequencing.
• To establish a combined CE-MS/MS and LC-MS/MS workflow for comprehensive antibody sequencing.

Main Methods:

• Utilized capillary electrophoresis tandem mass spectrometry (CE-MS/MS) with pH-mediated focusing.
• Employed less than 200 ng of tryptic digest sample for triplicated measurements.
• Applied a combined workflow integrating CE-MS/MS and LC-MS/MS.

Main Results:

• Achieved 100% sequence coverage and accuracy for the light chain of infliximab using CE-MS/MS.
• Obtained 96% coverage and 93% accuracy for the heavy chain of infliximab using CE-MS/MS.
• Demonstrated comparable or superior results for CE-MS/MS versus LC-MS/MS as standalone techniques.
• Successfully sequenced a novel antibody, anti-CD-176, using the combined workflow, providing its first proposed sequence.

Conclusions:

• CE-MS/MS is a viable and effective alternative for de novo sequencing of monoclonal antibodies.
• CE-MS/MS offers high efficiency and accuracy, even with limited sample amounts.
• The combined CE-MS/MS and LC-MS/MS approach enables comprehensive sequencing of novel antibodies.