Proteomics

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MS-based sequencing of the anti-FLAG-M2 antibody

ABSTRACT: We demonstrate a method for direct de novo sequencing of monoclonal IgG from the purified antibody products. The method uses a panel of multiple complementary proteases to generate suitable peptides for de novo sequencing by LC-MS/MS in a bottom-up fashion. Furthermore, we apply a dual fragmentation scheme, using both stepped high-energy collision dissociation (stepped HCD) and electron transfer high-energy collision dissociation (EThcD) on all peptide precursors. The method achieves full sequence coverage of the monoclonal antibody Herceptin, with an accuracy of 98% in the variable regions. We applied the method to sequence the widely used anti-FLAG-M2 mouse monoclonal antibody, which we successfully validated by remodeling a high-resolution crystal structure of the Fab and demonstrating binding to a FLAG-tagged target protein in Western blot analysis. The method thus offers robust and reliable sequences of monoclonal antibodies.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Homo Sapiens (human) Mus Musculus (mouse)

SUBMITTER: Joost Snijder  

LAB HEAD: Joost Snijder

PROVIDER: PXD023419 | Pride | 2021-01-11

REPOSITORIES: Pride

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Dataset's files

Source:
Action DRS
Peng2021_Herceptin_SupernovoPeptideOverview.csv Csv
Peng2021_Herceptin_aLP.raw Raw
Peng2021_Herceptin_aspN.raw Raw
Peng2021_Herceptin_chymo.raw Raw
Peng2021_Herceptin_elastase.raw Raw
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Publications

Mass Spectrometry-Based <i>De Novo</i> Sequencing of Monoclonal Antibodies Using Multiple Proteases and a Dual Fragmentation Scheme.

Peng Weiwei W   Pronker Matti F MF   Snijder Joost J  

Journal of proteome research 20210614 7

Antibody sequence information is crucial to understanding the structural basis for antigen binding and enables the use of antibodies as therapeutics and research tools. Here, we demonstrate a method for direct <i>de novo</i> sequencing of monoclonal IgG from the purified antibody products. The method uses a panel of multiple complementary proteases to generate suitable peptides for <i>de novo</i> sequencing by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in a bottom-up fashion. Furt  ...[more]

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