Fiche publication
Date publication
avril 2015
Auteurs
Membres identifiés du Cancéropôle Est :
Dr CIANFERANI Sarah
,
Dr VAN DORSSELAER Alain
Tous les auteurs :
Ayoub D, Bertaccini D, Diemer H, Wagner-Rousset E, Colas O, Cianferani S, Van Dorsselaer A, Beck A, Schaeffer-Reiss C
Lien Pubmed
Résumé
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.
Référence
Anal Chem. 2015 Apr 7;87(7):3784-90
