Standard-Free Absolute Quantitation of Antibody Deamidation Degradation and Host Cell Proteins by Coulometric Mass Spectrometry

Abstract

Proteomic absolute quantitation strategies mainly rely on the use of synthetic stable isotope-labeled peptides or proteins as internal standards, which are highly costly and timeconsuming to synthesize. To circumvent this limitation, we recently developed a coulometric mass spectrometry (CMS) approach for absolute quantitation of proteins without the use of standards, based on the electrochemical oxidation of oxidizable surrogate peptides, followed by mass spectrometry measurement of the peptide oxidation yield. Previously, CMS was only applied for single-protein quantitation. In this study, first, we demonstrated absolute quantitation of multiple proteins in a mixture (e.g., βlactoglobulin B, α-lactalbumin, and carbonic anhydrase) by CMS in one run, without using any standards. The CMS quantitation result was validated with a traditional isotope dilution method. Second, CMS can be used for absolute quantitation of a low-level target protein in a mixture; for instance, 500 ppm of PLBL2, a problematic host cell protein (HCP), in the presence of a highly abundant monoclonal antibody (mAb) was successfully quantified by CMS with no use of standards. Third, taking one step further, this study demonstrated the unprecedented quantitative analysis of deamidated peptide products arising from the mAb heavy chain deamidation reaction. In particular, absolute quantitation of the deamidation succinimide intermediate which had not been performed before due to the lack of standard was conducted by CMS, for the first time. Overall, our data suggest that CMS has potential utilities for quantitative proteomics and biotherapeutic drug discovery.

Influence of particle z‐potential and experimental procedure on protein corona formation and multicomponent aggregation

“…For protein digestion, 10 μL of trypsin (0.5 μg/μL Roche) was added and incubated with the sample at 37°C overnight (15–17 h). The digestion reaction was quenched with 1 μL of formic acid, and 10 μL of the resulting peptide solution was taken and diluted with 40 μL of acetonitrile/water/formic acid (ACN/H 2 O/FA, 50/50/0.1, v/v/v) for LC/MS analysis 25 …”

“…The digestion reaction was quenched with 1 μL of formic acid, and 10 μL of the resulting peptide solution was taken and diluted with 40 μL of acetonitrile/water/formic acid (ACN/H 2 O/FA, 50/50/0.1, v/v/v) for LC/MS analysis. 25 All LC/MS experiments were performed using ultraperformance liquid chromatography (UPLC, Waters, Milford, MA) coupled with a high-resolution Orbitrap Q Exactive mass spectrometer (Thermo Scientific, San Jose, CA). A reversed phase column (BEH C18, 2.1 mm  50 mm, 1.7 μm) was used for peptide separation of the protein digest mixture with a mobile phase flow rate of 200 μL/min.…”

Evolution of Vaccines Formulation to Tackle the Challenge of Anti-Microbial Resistant Pathogens

“…For example, the absolute quantification of protein content, asparagine deamidation, and host cell proteins in the same analytical session has recently been reported. A coulometric mass-spectrometry approach, based on the electrochemical properties of peptides, was used without the need for external standards or calibration curves [195]. Furthermore, the unprecedented sensitivity (up to the femtomolar range) and the dynamic range of linearity [196] achieved by the most recent generation of mass spectrometers can offer unique applications in the field of vaccine characterisation.…”

Standards-Free Absolute Quantitation of Oxidizable Glycopeptides by Coulometric Mass Spectrometry

“…Previously, we have established that the CMS method can be used for accurately quantifying small-molecule organic molecules (e.g., uric acid, N -nitrosamine), tyrosine- or tryptophan-containing peptides, and large-molecule proteins. − In this study, we applied the method for absolute quantitation of glycopeptides. By using our approach, we successfully quantified the absolute amounts of glycopeptides NYIVGQPSS­(β-GlcNAc)­TGNL and NYSVPSS­(β-GlcNAc)­TGNL.…”