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Characterization of the N-terminal Heterogeneities of Monoclonal Antibodies Using In-Gel Charge Derivatization of α-amines and LC-MS/MS

Characterization of the N-terminal Heterogeneities of Monoclonal Antibodies Using In-Gel Charge Derivatization of α-amines and LC-MS/MS

Daniel Ayoub, Diego Bertaccini, Hélène Diemer, Elsa Wagner-Rousset, Olivier Colas, Sarah Cianférani, Alain Van Dorsselaer, Alain Beck, Christine Schaeffer-Reiss

Anal Chem. 2015 Apr 7;87(7):3784-90.

PMID: 25769014

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.

Chemicals Related in the Paper:

Catalog Number	Product Name	Structure	CAS Number	Price
AP226409581	(N-Succinimidyloxycarbonylmethyl)tris(2,4,6-trimethoxyphenyl)phosphonium bromide		226409-58-1	Price

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