We apologize for Proteopedia being slow to respond. For the past two years, a new implementation of Proteopedia has been being built. Soon, it will replace this 18-year old system. All existing content will be moved to the new system at a date that will be announced here.

7bg1

From Proteopedia

Jump to: navigation, search

Structure of anti-FLAG M2 Fab domain remodeled based on proteomic sequencing

Structural highlights

7bg1 is a 2 chain structure with sequence from Mus musculus. Full crystallographic information is available from OCA. For a guided tour on the structure components use FirstGlance.
Method:	X-ray diffraction, Resolution 1.86Å
Ligands:	,
Resources:	FirstGlance, OCA, PDBe, RCSB, PDBsum, ProSAT

Publication Abstract from PubMed

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 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 liquid chromatography-tandem mass spectrometry (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 99% 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.

Mass Spectrometry-Based De Novo Sequencing of Monoclonal Antibodies Using Multiple Proteases and a Dual Fragmentation Scheme.,Peng W, Pronker MF, Snijder J J Proteome Res. 2021 Jul 2;20(7):3559-3566. doi: 10.1021/acs.jproteome.1c00169. , Epub 2021 Jun 14. PMID:34121409^[1]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.

References

↑ Peng W, Pronker MF, Snijder J. Mass Spectrometry-Based De Novo Sequencing of Monoclonal Antibodies Using Multiple Proteases and a Dual Fragmentation Scheme. J Proteome Res. 2021 Jul 2;20(7):3559-3566. PMID:34121409 doi:10.1021/acs.jproteome.1c00169