Characterization of the N-terminal heterogeneities of monoclonal antibodies using in-gel charge derivatization of α-amines and LC-MS/MS

Analytical Chemistry
Daniel AyoubChristine Schaeffer-Reiss

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.

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Citations

Mar 8, 2018·MAbs·Alexandre AmbrogellyHongcheng Liu
Aug 1, 2015·Proteomics·Jean Armengaud
Dec 15, 2015·Proteomics·Stéphane TrevisiolBruno Domon
Nov 10, 2015·Proteomics. Clinical Applications·Yuting CongJingkai Gu
Sep 24, 2019·Antibodies·Alain Beck, Hongcheng Liu
Oct 23, 2016·Biotechnology Progress·Hongcheng LiuAlyssa Neill
Nov 6, 2018·Analytical Chemistry·Tianjiao HuangJames L Edwards
Oct 8, 2021·Analytical Chemistry·Seonjeong LeeCheolju Lee

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