Analysis of Monoclonal Antibody Sequence and Post-translational Modifications by Time-controlled Proteolysis and Tandem Mass Spectrometry

Abstract

Methodology for sequence analysis of ϳ150 kDa monoclonal antibodies (mAb), including location of post-translational modifications and disulfide bonds, is described. Limited digestion of fully denatured (reduced and alkylated) antibody was accomplished in seconds by flowing a sample in 8 M urea at a controlled flow rate through a micro column reactor containing immobilized aspergillopepsin I. The resulting product mixture containing 3-9 kDa peptides was then fractionated by capillary column liquid chromatography and analyzed on-line by both electron-transfer dissociation and collisionally activated dissociation mass spectrometry (MS). This approach enabled identification of peptides that cover the complete sequence of a murine mAb. With customized tandem MS and ProSightPC Biomarker search, we verified 95% amino acid residues of this mAb and identified numerous posttranslational modifications (oxidized methionine, pyroglutamylation, deamidation of Asn, and several forms of Nlinked glycosylation). For disulfide bond location, native mAb is subjected to the same procedure but with longer digestion times controlled by sample flow rate through the micro column reactor. Release of disulfide containing peptides from accessible regions of the folded antibody occurs with short digestion times. Release of those in the interior of the molecule requires longer digestion times. The identity of two peptides connected by a disulfide bond is determined using a combination of electron-transfer dissociation and ion-ion proton transfer chemistry to read the two N-terminal and two C-terminal sequences of the connected peptides. Molecular & Cellular Proteomics

Overlooked Benefits of using Polyclonal Antibodies

“…For instance, epitopes on targets that represent a small change such as a single amino acid polymorphism [34,35] or a post-translational modification [36,37] (e.g.,phosphorylation, methylation, hydroxylation), may be better targeted using a mAb. Often these types of targets or changes are too small to elicit a robust pAb immune response and in addition increase the risk of nonspecific antibody reactivity within the pAb mixture.…”

The Role of Electron Transfer Dissociation in Modern Proteomics

“… 305 Additionally, multiple fills of ETD-PTR product ions can be collected prior to mass analysis in this approach, considerably enhancing observed ion current and product ion S/N without the need for time-consuming averaging of data from multiple mass measurements. The ETD/PTR technique proved extremely valuable in middle down analyses of antibodies, 243 and they further extended its utility in a recent top-down characterization of histones by incorporating parallel ion parking 35 , 306 during the PTR reactions to specifically control reaction of precursor ions to remain in a targeted product m / z range. 237 The value of these strategies is clear for analysis of large peptides and proteins, and we expect this technology will be widely implemented in top-down experiments in coming years.…”

Front-End Electron Transfer Dissociation Coupled to a 21 Tesla FT-ICR Mass Spectrometer for Intact Protein Sequence Analysis

“…Electron transfer dissociation (ETD) for top-down- or middle-down-size polypeptides has produced unprecedented sequence coverage [21–23]. Fragment ions produced by ETD retain labile post-translational modifications (PTMs) [24], and thus enable identification of their sequence location(s).…”