title: Characterization of the N-terminal heterogeneities of monoclonal antibodies using in-gel charge derivatization of α-amines and LC-MS/MS.
abstract: The bioproduction of recombinant monoclonal antibodies results in complex mixtures of a main isoform and numerous macro- and microvariants. Monoclonal antibodies therefore present different levels of heterogeneities ranging from primary sequence variants to post-translational modifications. Among these heterogeneities, the truncation and fragmentation of the primary amino-acid sequence result in shorter or cleaved polypeptide chains while the incomplete processing of the signal peptide produces N-terminal elongated polypeptide chains. Here, we present an in-gel protein N-terminal chemical derivatization method using (N-succinimidyloxycarbonylmethyl)-tris(2,4,6-trimethoxyphenyl)phosphonium bromide (TMPP). This chemical tag enhances the detection by mass spectrometry of the N-terminal positions of proteins and allows their unambiguous assignment without altering the identification of internal digestion peptides. This method adds just one step to the classical peptide mapping workflow. Using this in-gel N-TOP (N-terminal oriented proteomics) strategy, the N-terminal sequence heterogeneities of several monoclonal antibodies, among which are minor unexpected proteoforms, were successfully detected and characterized.
authors.affiliation: †Centre d'Immunologie Pierre Fabre (CIPF), 5 Av. Napoléon III, BP 60497, 74164 Saint-Julien-en-Genevois, France.
authors.forename: Daniel
authors.initials: D
authors.lastname: Ayoub
authors.affiliation: ‡BioOrganic Mass Spectrometry Laboratory (LSMBO), IPHC, Université de Strasbourg, 25 rue Becquerel, 67087 Strasbourg, France.|∥IPHC, CNRS, UMR7178, 67087 Strasbourg, France.
authors.forename: Diego
authors.initials: D
authors.lastname: Bertaccini
authors.affiliation: ‡BioOrganic Mass Spectrometry Laboratory (LSMBO), IPHC, Université de Strasbourg, 25 rue Becquerel, 67087 Strasbourg, France.|∥IPHC, CNRS, UMR7178, 67087 Strasbourg, France.
authors.forename: Hélène
authors.initials: H
authors.lastname: Diemer
authors.affiliation: †Centre d'Immunologie Pierre Fabre (CIPF), 5 Av. Napoléon III, BP 60497, 74164 Saint-Julien-en-Genevois, France.
authors.forename: Elsa
authors.initials: E
authors.lastname: Wagner-Rousset
authors.affiliation: †Centre d'Immunologie Pierre Fabre (CIPF), 5 Av. Napoléon III, BP 60497, 74164 Saint-Julien-en-Genevois, France.
authors.forename: Olivier
authors.initials: O
authors.lastname: Colas
authors.affiliation: ‡BioOrganic Mass Spectrometry Laboratory (LSMBO), IPHC, Université de Strasbourg, 25 rue Becquerel, 67087 Strasbourg, France.|∥IPHC, CNRS, UMR7178, 67087 Strasbourg, France.
authors.forename: Sarah
authors.initials: S
authors.lastname: Cianférani
authors.affiliation: ‡BioOrganic Mass Spectrometry Laboratory (LSMBO), IPHC, Université de Strasbourg, 25 rue Becquerel, 67087 Strasbourg, France.|∥IPHC, CNRS, UMR7178, 67087 Strasbourg, France.
authors.forename: Alain
authors.initials: A
authors.lastname: Van Dorsselaer
authors.affiliation: †Centre d'Immunologie Pierre Fabre (CIPF), 5 Av. Napoléon III, BP 60497, 74164 Saint-Julien-en-Genevois, France.
authors.forename: Alain
authors.initials: A
authors.lastname: Beck
authors.affiliation: ‡BioOrganic Mass Spectrometry Laboratory (LSMBO), IPHC, Université de Strasbourg, 25 rue Becquerel, 67087 Strasbourg, France.|∥IPHC, CNRS, UMR7178, 67087 Strasbourg, France.
authors.forename: Christine
authors.initials: C
authors.lastname: Schaeffer-Reiss
mesh_terms: D000577:Amides / Q000737:chemistry*; D000911:Antibodies, Monoclonal / Q000032:analysis* / Q000737:chemistry*; D002460:Cell Line; D002853:Chromatography, Liquid; D005782:Gels / Q000737:chemistry*; D006801:Humans; D053719:Tandem Mass Spectrometry
chemical_list: D000577:Amides; D000911:Antibodies, Monoclonal; D005782:Gels